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OXIDATIVE STRESS AND DISEASE 

Series Editors 

LESTER PACKER, PH.D. 

ENRIQUE CADENAS, M.D., PH.D. 

University of Southern California School of Pharmacy 

Los Angeles, California 

1. Oxidative Stress in Cancer, AIDS, and Neurodegenerative 

Diseases, edited by Luc Montagnier, René Olivier, and 

Catherine Pasquier 

2. Understanding the Process of Aging: The Roles of 

Mitochondria, Free Radicals, and Antioxidants, edited by 

Enrique Cadenas and Lester Packer 

3. Redox Regulation of Cell Signaling and Its Clinical Application, 

edited by Lester Packer and Junji Yodoi 

4. Antioxidants in Diabetes Management, edited by Lester Packer, 

Peter Rösen, Hans J. Tritschler, George L. King, 

and Angelo Azzi 

5. Free Radicals in Brain Pathophysiology, edited by 

Giuseppe Poli, Enrique Cadenas, and Lester Packer 

6. Nutraceuticals in Health and Disease Prevention, edited by 

Klaus Krämer, Peter-Paul Hoppe, and Lester Packer 

7. Environmental Stressors in Health and Disease, edited by 

Jürgen Fuchs and Lester Packer 

8. Handbook of Antioxidants: Second Edition, Revised 

and Expanded, edited by Enrique Cadenas and Lester Packer 

9. Flavonoids in Health and Disease: Second Edition, Revised 

and Expanded, edited by Catherine A. Rice-Evans 

and Lester Packer 

10. Redox–Genome Interactions in Health and Disease, edited by 

Jürgen Fuchs, Maurizio Podda, and Lester Packer 

11. Thiamine: Catalytic Mechanisms in Normal and Disease States, 

edited by Frank Jordan and Mulchand S. Patel 

12. Phytochemicals in Health and Disease, edited by Yongping Bao 

and Roger Fenwick 

13. Carotenoids in Health and Disease, edited by Norman I. Krinsky, 

Susan T. Mayne, and Helmut Sies

14. Herbal and Traditional Medicine: Molecular Aspects of Health, 

edited by Lester Packer, Choon Nam Ong, and Barry Halliwell 

15. Nutrients and Cell Signaling, edited by Janos Zempleni 

and Krishnamurti Dakshinamurti 

16. Mitochondria in Health and Disease, edited by 

Carolyn D. Berdanier 

17. Nutrigenomics, edited by Gerald Rimbach, Jürgen Fuchs, 

and Lester Packer 

18. Oxidative Stress, Inflammation, and Health, edited by 

Young-Joon Surh and Lester Packer 

19. Nitric Oxide, Cell Signaling, and Gene Expression, edited by 

Santiago Lamas and Enrique Cadenas 

20. Resveratrol in Health and Disease, edited by 

Bharat B. Aggarwal and Shishir Shishodia 

21. Oxidative Stress and Age-Related Neurodegeneration, 

edited by Yuan Luo and Lester Packer 

22. Molecular Interventions in Lifestyle-Related Diseases, edited by 

Midori Hiramatsu, Toshikazu Yoshikawa, and Lester Packer 

23. Oxidative Stress and Inflammatory Mechanisms in Obesity, 

Diabetes, and the Metabolic Syndrome, edited by 

Lester Packer and Helmut Sies

Oxidative Stress 

and Inflammatory 

Mechanisms in Obesity, 

Diabetes, and the 

Metabolic Syndrome 

edited by 

Lester Packer 

Helmut Sies 

Boca Raton London New York 

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Library of Congress Cataloging-in-Publication Data 

Oxidative stress and inflammatory mechanisms in obesity, diabetes, and the 

metabolic syndrome / edited by Lester Packer and Helmut Sies. 

p. ; cm. -- (Oxidative stress and disease ; 22) 

“A CRC title.” 

Includes bibliographical references and index. 

ISBN-13: 978-1-4200-4378-5 (hardcover : alk. paper) 

ISBN-10: 1-4200-4378-1 (hardcover : alk. paper) 

1. Oxidative stress. 2. Metabolic syndrome. 3. Obesity. 4. Diabetes. I. Packer, 

Lester. II. Sies, H. (Helmut), 1942- III. Series. 

[DNLM: 1. Metabolic Syndrome X--physiopathology. 2. Diabetes Mellitus, Type 

2--physiopathology. 3. Inflammation--physiopathology. 4. Obesity--physiopathology. 

5. Oxidative Stress--physiology. W1 OX626 v.22 2007 / WK 820 O98 2007] 

QP177.O935 2007 

616.3’9--dc22 2007005664 

Visit the Taylor & Francis Web site at 

http://www.taylorandfrancis.com 

and the CRC Press Web site at 

http://www.crcpress.com

Table of Contents 

Series Preface.......................................................................................................xi 

Preface.................................................................................................................xv 

About the Editors............................................................................................. xvii 

Contributors........................................................................................................xix 

SECTION I Oxidative Stress, Metabolic Syndrome, 

Obesity, Diabetes, and Uncoupling 

Proteins 

Chapter 1 

The Metabolic Syndrome Defined........................................................................3 

Neil J. Stone and Jennifer Berliner 

Chapter 2 

The Metabolic Syndrome: The Question of Balance between the 

Pro-Inflammatory Effect of Macronutrients and the Anti-Inflammatory 

Effect of Insulin ..................................................................................................15 

Paresh Dandona, Ajay Chaudhuri, Priya Mohanty, and Husam Ghanim 

Chapter 3 

The Role of Oxidative Stress in Diseases Associated with 

Overweight and Obesity .....................................................................................33 

Ginger L. Milne, Ling Gao, Joshua D. Brooks, and Jason D. Morrow 

Chapter 4 

Metabolic Syndrome Due to Early Life Nutritional Modifications...................47 

Malathi Srinivasan, Paul Mitrani, and Mulchand S. Patel 

Chapter 5 

Oxidative Stress and Antioxidants in the Perinatal Period ................................71 

Hiromichi Shoji, Yuichiro Yamashiro, and Berthold Koletzko 

vii

viii Oxidative Stress and Inflammatory Mechanisms 

Chapter 6 

Maternal Obesity, Glucose Intolerance, and Inflammation in Pregnancy .........93 

Janet C. King 

Chapter 7 

Obesity, Nutrigenomics, Metabolic Syndrome, and Type 2 Diabetes.............107 

David Heber 

Chapter 8 

Post-Prandial Endothelial Dysfunction, Oxidative Stress, and 

Inflammation in Type 2 Diabetes .....................................................................123 

Antonio Ceriello 

Chapter 9 

Obesity and Inflammation: Implications for Atherosclerosis ..........................139 

John Alan Farmer 

Chapter 10 

Oligomeric Composition of Adiponectin and Obesity.....................................167 

T. Bobbert and Joachim Spranger 

Chapter 11 

Insulin-Stimulated Reactive Oxygen Species and Insulin 

Signal Transduction...........................................................................................177 

Barry J. Goldstein, Kalyankar Mahadev, and Xiangdong Wu 

Chapter 12 

Intracellular Signaling Pathways and Peroxisome Proliferator-Activated 

Receptors in Vascular Health in Hypertension and in Diabetes ......................195 

Farhad Amiri, Karim Benkirane, and Ernesto L. Schiffrin 

Chapter 13 

Role of Uncoupling Protein 2 in Pancreatic β Cell Function: 

Secretion and Survival ......................................................................................211 

Jingyu Diao, Catherine B. Chan, and Michael B. Wheeler

Table of Contents ix 

SECTION II Influence of Dietary Factors, 

Micronutrients, and Metabolism 

Chapter 14 

Nutritional Modulation of Inflammation in Metabolic Syndrome...................227 

Uma Singh, Sridevi Devaraj, and Ishwarlal Jialal 

Chapter 15 

Dietary Fatty Acids and Metabolic Syndrome.................................................243 

Helen M. Roche 

Chapter 16 

Lipid-Induced Death of Macrophages: Implication for 

Destabilization of Atherosclerotic Plaques.......................................................251 

Oren Tirosh and Anna Aronis 

Chapter 17 

α-Lipoic Acid Prevents Diabetes Mellitus and Endothelial 

Dysfunction in Diabetes-Prone Obese Rats .....................................................261 

Woo Je Lee, Ki-Up Lee, and Joong-Yeol Park 

Chapter 18 

Lipoic Acid Blocks Obesity through Reduced Food Intake, Enhanced 

Energy Expenditure, and Inhibited Adipocyte Differentiation ........................273 

Jong-Min Park and An-Sik Chung 

Chapter 19 

Effects of Conjugated Linoleic Acid and Lipoic Acid on Insulin 

Action in Insulin-Resistant Obese Zucker Rats ...............................................289 

Erik J. Henriksen 

Chapter 20 

Trivalent Chromium Supplementation Inhibits Oxidative Stress, 

Protein Glycosylation, and Vascular Inflammation in High 

Glucose-Exposed Human Erythrocytes and Monocytes ..................................301 

Sushil K. Jain 

Index .................................................................................................................315

Series Preface 

OXYGEN BIOLOGY AND MEDICINE 

Through evolution, oxygen — itself a free radical — was chosen as the terminal 

electron acceptor for respiration. The two unpaired electrons of oxygen spin in 

the same direction; thus, oxygen is a biradical. Other oxygen-derived free radicals 

such as superoxide anion or hydroxyl radicals formed during metabolism or by 

ionizing radiation are stronger oxidants, i.e., endowed with higher chemical 

reactivities. Oxygen-derived free radicals are generated during metabolism and 

energy production in the body and are involved in regulation of signal transduction 

and gene expression, activation of receptors and nuclear transcription factors, 

oxidative damage to cell components, antimicrobial and cytotoxic actions of 

immune system cells, as well as in aging and age-related degenerative diseases. 

Conversely, cells conserve antioxidant mechanisms to counteract the effects of 

oxidants; these antioxidants may remove oxidants either in a highly specific 

manner (for example, by superoxide dismutases) or in a less specific manner (for 

example, through small molecules such as vitamin E, vitamin C, and glutathione). 

Oxidative stress as classically defined is an imbalance between oxidants and 

antioxidants. Overwhelming evidence indicates that oxidative stress can lead to 

cell and tissue injury. However, the same free radicals that are generated during 

oxidative stress are produced during normal metabolism and, as a corollary, are 

involved in both human health and disease. 

UNDERSTANDING OXIDATIVE STRESS 

In recent years, the research disciplines interested in oxidative stress have grown 

and enormously increased our knowledge of the importance of the cell redox 

status and the recognition of oxidative stress as a process with implications for 

many pathophysiological states. From this multi- and inter-disciplinary interest 

in oxidative stress emerges a concept that attests to the vast consequences of 

the complex and dynamic interplay of oxidants and antioxidants in cellular and 

tissue settings. Consequently, our view of oxidative stress is growing in scope 

and new future directions. Likewise, the term reactive oxygen species — adopted 

at some stage in order to highlight non-radical oxidants such as H 2O 2 and 1 O 2 

— now fails to reflect the rich variety of other reactive species in free radical 

biology and medicine encompassing nitrogen- , sulfur- , oxygen- , and carboncentered 

radicals. With the discovery of nitric oxide, nitrogen-centered radicals 

xi

xii Oxidative Stress and Inflammatory Mechanisms 

gathered momentum and have matured into an area of enormous importance in 

biology and medicine. Nitric oxide or nitrogen monoxide (NO), a free radical 

generated in a variety of cell types by nitric oxide synthases (NOSs), is involved 

in a wide array of physiological and pathophysiological phenomena such as 

vasodilation, neuronal signaling, and inflammation. Of great importance is the 

radical–radical reaction of nitric oxide with superoxide anion. This is among 

the most rapid non-enzymatic reactions in biology (well over the diffusioncontrolled 

limits) and yields the potent non-radical oxidant, peroxynitrite. The 

involvement of this species in tissue injury through oxidation and nitration 

reactions is well documented. 

Virtually all diseases thus far examined involve free radicals. In most cases, 

free radicals are secondary to the disease process, but in some instances causality 

is established by free radicals. Thus, there is a delicate balance between oxidants 

and antioxidants in health and disease. Their proper balance is essential for 

ensuring healthy aging. 

Both reactive oxygen and nitrogen species are involved in the redox regulation 

of cell functions. Oxidative stress is increasingly viewed as a major upstream 

component in the signaling cascade involved in inflammatory responses, stimulation 

of cell adhesion molecules, and chemoattractant production and as an early 

component in age-related neurodegenerative disorders such as Alzheimer’s, Parkinson’s, 

and Huntington’s diseases, and amyotrophic lateral sclerosis. Hydrogen 

peroxide is probably the most important redox signaling molecule that, among 

others, can activate NFκB, Nrf2, and other universal transcription factors. Increasing 

steady-state levels of hydrogen peroxide have been linked to a cell’s redox 

status with clear involvement in adaptation, proliferation, differentiation, apoptosis, 

and necrosis. 

The identification of oxidants in regulation of redox cell signaling and gene 

expression was a significant breakthrough in the field of oxidative stress: the 

classical definition of oxidative stress as an imbalance between the production 

of oxidants and the occurrence of cell antioxidant defenses proposed by Sies 

in 1985 now seems to provide a limited concept of oxidative stress, but it 

emphasizes the significance of cell redox status. Because individual signaling 

and control events occur through discrete redox pathways rather than through 

global balances, a new definition of oxidative stress was advanced by Dean P. 

Jones (Antioxidants & Redox Signaling [2006]) as a disruption of redox signaling 

and control that recognizes the occurrence of compartmentalized cellular 

redox circuits. Recognition of discrete thiol redox circuits led Jones to provide 

this new definition of oxidative stress. Measurements of GSH/GSSG, cysteine/cystine, 

or thioredoxin reduced/thioredoxin oxidized provide a quantitative definition 

of oxidative stress. Redox status is thus dependent on the degree to which 

tissue-specific cell components are in the oxidized state. 

In general, the reducing environments inside cells help to prevent oxidative 

damage. In this reducing environment, disulfide bonds (S–S) do not spontaneously 

form because sulfhydryl groups are maintained in the reduced state (SH), thus 

preventing protein misfolding or aggregation. The reducing environment is

Series Preface xiii 

maintained by metabolism and by the enzymes involved in maintenance of 

thiol/disulfide balance and substances such as glutathione, thioredoxin, vitamins 

E and C, and enzymes such as superoxide dismutases, catalase, and the seleniumdependent 

glutathione reductase and glutathione and thioredoxin-dependent 

hydroperoxidases (periredoxins) that serve to remove reactive oxygen species 

(hydroperoxides). Also of importance is the existence of many tissue- and cell 

compartment-specific isoforms of antioxidant enzymes and proteins. 

Compelling support for the involvement of free radicals in disease development 

originates from epidemiological studies showing that enhanced antioxidant 

status is associated with reduced risk of several diseases. Of great significance 

is the role that micronutrients play in modulation of redox cell signaling; this 

establishes a strong linking of diet and health and disease centered on the abilities 

of micronutrients to regulate redox cell signaling and modify gene expression. 

These concepts are anticipated to serve as platforms for the development of 

tissue-specific therapeutics tailored to discrete, compartmentalized redox circuits. 

This, in essence, dictates principles of drug development-guided knowledge of 

mechanisms of oxidative stress. Hence, successful interventions will take advantage 

of new knowledge of compartmentalized redox control and free radical 

scavenging. 

OXIDATIVE STRESS IN HEALTH AND DISEASE 

Oxidative stress is an underlying factor in health and disease. In this series of 

books, the importance of oxidative stress and diseases associated with organ 

systems of the body is highlighted by exploring the scientific evidence and clinical 

applications of this knowledge. This series is intended for researchers in the basic 

biomedical sciences and clinicians. The potential of such knowledge for healthy 

aging and disease prevention warrants further knowledge about how oxidants and 

antioxidants modulate cell and tissue function. 

Lester Packer 

Enrique Cadenas

Preface 

Metabolic syndrome is a multicomponent disorder characterized by obesity, insulin 

resistance, dyslipidemia, and hypertension that is associated with the risks of 

type 2 diabetes mellitus and cardiovascular disease. Obesity plays a central role 

and is a principal causative factor in the progression of metabolic syndrome 

leading to type 2 diabetes mellitus and cardiovascular disease. Increased systemic 

oxidative stress may be an important mechanism by which obesity increases the 

incidence of atherosclerotic cardiovascular disease, the progression of which 

entails oxidative (up-regulation of oxidative enzymes) and inflammatory 

(increased production of tumor necrosis factors and other cytokines) components. 

The significance of inflammatory processes in accumulated fat appears to be an 

early initiator of metabolic syndrome, thus strengthening an association with 

oxidative stress and supporting the development of drugs targeted at ameliorating 

cellular redox status. Likewise, the more active angiotensin system in obesity 

may contribute to expanded oxidative stress that serves as a key signaling event 

in vascular remodeling. 

A greater understanding of the molecular mechanisms that underlie inflammation 

and oxidative stress with implications for metabolic stress must be 

achieved so that evidence-based nutritional and pharmacological therapies can 

be developed to attenuate the impacts of obesity-induced insulin resistance and 

ensuing metabolic syndrome. 

The chapters in this book report cutting-edge research exploring the intracellular 

events mediating or preventing oxidative stress and pro-inflammatory processes 

in obesity and type 2 diabetes as well as the molecular mechanisms inherent 

in the progression of metabolic stress, phenotypic perspectives, dietary factors, 

and micronutrients. They attempt to provide perspectives on the different components 

of metabolic stress and obesity and their associations with oxidative stress 

and inflammation. Pharmacological interventions in disease management are not 

emphasized and are covered elsewhere. 

The editors gratefully appreciate the initial stimulus for this book — a workshop 

on obesity, oxidative stress related to metabolic syndrome, uncoupling 

proteins, and micronutrient action conducted March 15 through 18, 2006 as part 

of the Twelfth Annual Meeting of the Oxygen Club of California at Santa Barbara. 

We would like to acknowledge the sponsorship of that workshop by the Human 

Nutrition Group of BASF, Ludwigshafen, Germany, and, in particular, the input 

and help of Dr. Ute Obermüller-Jevic. 

xv

About the Editors 

Lester Packer received his PhD in microbiology and biochemistry from Yale 

University. For many years he was professor and senior researcher at the University 

of California at Berkeley. Currently he is an adjunct professor in the 

Department of Pharmacology and Pharmaceutical Sciences at the University of 

Southern California. Recently, he was appointed distinguished professor at the 

Institute of Nutritional Sciences of the Chinese Academy of Sciences, Shanghai, 

China. His research interests are related to the molecular, cellular, and physiological 

role of oxidants, free radicals, antioxidants, and redox regulation in health 

and disease. 

Professor Packer is the recipient of numerous scientific achievement awards 

including three honorary doctoral degrees. He has served as president of the 

International Society of Free Radical Research (SRRRI), president of the Oxygen 

Club of California (OCC), and vice-president of UNESCO—the United Nations 

Global Network on Molecular and Cell Biology (MCBN). 

Helmut Sies earned an MD from the University of Munich, Germany, and an 

honorary PhD from the University of Buenos Aires, Argentina. He is a professor 

and chairman at the Institute of Biochemistry and Molecular Biology I at Heinrich 

Heine University at Dusseldorf, Germany. He served as a visiting professor at 

the University of California (Berkeley) and is an adjunct professor at the University 

of Southern California. He is a fellow of the National Foundation for 

Cancer Research based in Bethesda, Maryland and a fellow of the Royal College 

of Physicians of London. 

Dr. Sies has been president of the Society for Free Radical Research International 

and is also the president of the Oxygen Club of California. His research 

interests focus on the field of oxidative stress, oxidants, and antioxidants. 

xvii

Contributors 

Farhad Amiri, PhD 

Lady Davis Institute for Medical 

Research 

Sir Mortimer B. Davis-Jewish General 

Hospital 

McGill University 

Montreal, Canada 

Anna Aronis 

School of Nutritional Sciences 

Institute of Biochemistry, Food 

Science, and Nutrition 

The Hebrew University of Jerusalem 

Rehovot, Israel 

Karim Benkirane, PhD 


Research 


Hospital 



Jennifer Berliner, MD 

Cardiology Section 

Feinberg School of Medicine 

Northwestern University 

Chicago, Illinois 

T. Bobbert 

Abteilung fur Endokrinologie, 

Diabetes und Ernahrung 

Charite Universitatsmedizin 

Berlin, Germany 

Joshua D. Brooks 

Division of Clinical Pharmacology 

Vanderbilt University Medical 

Center 

Nashville, Tennessee 

Antonio Ceriello, MD 

Warwick Medical School 

University of Warwick 

Coventry, United Kingdom 

Catherine B. Chan 

Department of Biomedical 

Sciences 

Atlantic Veterinary College 

University of Prince Edward Island 

Charlottetown, Canada 

Ajay Chaudhuri, MD 

School of Medicine and Biomedical 

Sciences 

State University of New York 

and 

Kaleida Health 

Buffalo, New York 

An-Sik Chung 

Department of Biological 

Sciences 

Korea Advanced Institute of Science 

and Technology 

Daejeon, Republic of Korea 

xix

xx Oxidative Stress and Inflammatory Mechanisms 

Paresh Dandona, MD, PhD 


Sciences 


and 



Sridevi Devaraj 

Laboratory for Atherosclerosis and 

Metabolic Research 

University of California at Davis 

Medical Center 

Sacramento, California 

Jingyu Diao 

Department of Physiology 

Faculty of Medicine 

University of Toronto 

Toronto, Canada 


Baylor College of Medicine 

Houston, Texas 

Ling Gao 


Vanderbilt University Medical Center 


Husam Ghanim, PhD 


Sciences 


and 



Barry J. Goldstein 

Dorrance Hamilton Research 

Laboratories 

Jefferson Medical College 

Thomas Jefferson University 

Philadelphia, Pennsylvania 

David Heber, MD, PhD, FACP, 

FACN 

Center for Human Nutrition 

David Geffen School of Medicine 

University of California 

Los Angeles, California 



College of Medicine 

University of Arizona 

Tucson, Arizona 


Department of Pediatrics 

Health Sciences Center 

Louisiana State University 

Shreveport, Louisiana 

Ishwarlal Jialal, MD, PhD 






Janet C. King 

Children’s Hospital 

Oakland Research Institute 

Oakland, California 

Berthold Koletzko, MD 

Division of Metabolic Disorders and 

Nutrition 

Dr. von Hauner Children’s Hospital 

Ludwig Maximilians University 

Munich, Germany 

Ki-Up Lee 

Department of Internal Medicine 

University of Ulsan College of 

Medicine 

Seoul, Republic of Korea

Contributors xxi 

Woo Je Lee 


Inje University College of Medicine 

Seoul, Republic of Korea 

Kalyankar Mahadev 


Laboratories 




Ginger L. Milne 



Center 


Paul Mitrani 

Department of Biochemistry 


Sciences 



Priya Mohanty, MD 


Sciences 


and 



Jason D. Morrow, MD 



Center 


Jong-Min Park 

Department of Biological Sciences 

Korea Advanced Institute of Science 

and Technology 

Daejeon, Republic of Korea 

Joong-Yeol Park 


University of Ulsan College of 

Medicine 

Seoul, Republic of Korea 

Mulchand S. Patel 



Sciences 




Institute of Molecular Medicine 

Trinity Health Sciences Centre 

St. James Hospital 

Dublin, Republic of Ireland 

Ernesto L. Schiffrin, MD, PhD, 

FRSC, FRCPC, FACP 


Research 


Hospital 



Hiromichi Shoji 


Juntendo University School of 

Medicine 

Tokyo, Japan 

and 

Division of Metabolic Disorders and 

Nutrition 

Dr. von Hauner Children’s 

Hospital 

Ludwig Maximilians University 

Munich, Germany

xxii Oxidative Stress and Inflammatory Mechanisms 

Uma Singh 






Joachim Spranger 

Department of Clinical Nutrition 

German Institute of Human Nutrition 

Potsdam-Rehbruecke 

Nuthetal, Germany 

Malathi Srinivasan 



Sciences 



Neil J. Stone, MD 

Cardiology Section 

Feinberg School of Medicine 

Northwestern University 

Chicago, Illinois 

Oren Tirosh 

School of Nutritional Sciences 

Institute of Biochemistry, Food 

Science, and Nutrition 

The Hebrew University of Jerusalem 

Rehovot, Israel 

Michael B. Wheeler 


Faculty of Medicine 

University of Toronto 

Toronto, Canada 

Xiangdong Wu 


Laboratories 




Yuichiro Yamashiro 


Juntendo University School of 

Medicine 

Tokyo, Japan

Section I 

Oxidative Stress, Metabolic 

Syndrome, Obesity, Diabetes, 

and Uncoupling Proteins

1 

CONTENTS 

The Metabolic 

Syndrome Defined 

Neil J. Stone and Jennifer Berliner 

Rationale for Defining Metabolic Syndrome .......................................................3 

Metabolic Syndrome Definitions..........................................................................5 

World Health Organization (WHO) Definition...........................................5 

ATP III Initial Report ..................................................................................5 

American Association of Clinical Endocrinologists Guidelines ................5 

European Group for Study of Insulin Resistance Syndrome .....................7 

Revised ATP III Guidelines.........................................................................7 

International Diabetes Federation (IDF) Definition ...................................8 

Is Metabolic Syndrome Useful and Which Definition Should be Used?............9 

References ...........................................................................................................13 

RATIONALE FOR DEFINING METABOLIC SYNDROME 

In the past decade, sedentary lifestyles, atherogenic high calorie diets, and weight 

gains have characterized adolescents and adults in the United States and in many 

countries across the globe. 1,2 Indeed, a recent report 2 estimated the prevalences 

of overweight and obese people in the U.S. above 60 and 30%, respectively. This 

is not a unique burden for the U.S., but reflects a worldwide trend demonstrating 

an increased prevalence in metabolic risk factors 3,4 including visceral obesity, 

insulin resistance, dyslipidemia with abnormal values for triglycerides and high 

density lipoprotein cholesterol (HDL-c), hypertension, and (if measured) prothrombotic 

and inflammatory markers. 

Although metabolic risk factors track with body mass index (BMI) calculated 

as weight/height squared, this has not proven the best correlate of coronary heart 

disease (CHD) events. More than 50 years ago, a French investigator drew 

attention to fat distribution in a graphic way showing the difference between those 

with android or abdominal patterns and those with gynecoid or female patterns 

of fat distribution. 5 In 2004, the global INTERHEART project demonstrated that 

the measure of obesity most directly related to myocardial infarction (MI) was 

3

4 Oxidative Stress and Inflammatory Mechanisms 

not BMI, but a measure of body fatness, the waist:hip ratio. 6 Although not as 

good as the waist:hip ratio, waist measurements were more predictive than BMI. 

In addition to the appreciation that weight gain and obesity are increasing 

global problems and that the location of body fat has both prognostic and therapeutic 

importance is the recognition of atherogenic risk factor clustering. This 

concept was noted by Framingham investigators who found it was common in 

both men and women, worsened with weight gain, and appreciably increased the 

risk of CHD. 7 The odds ratios for individuals with this risk factor clustering was 

more than two-fold for men and more than six-fold for women. Subsequent 

studies identified factors that can intensify these metabolic abnormalities. They 

include aging (higher prevalence at higher age), ethnic subgroups, endocrine 

dysfunction (increased prevalence in polycystic ovary syndrome), and physical 

inactivity. 8 The distinction from those unaffected is not trivial. Characteristic 

clinical features of risk factor clustering identify individuals who are more likely 

to become diabetic and/or experience a cardiac event. This spurred interest in 

viewing this constellation of clustered metabolic risk factors as a syndrome. It is 

important to recognize that metabolic syndrome is not a disease entity with a 

single etiologic cause. Nonetheless, the metabolic syndrome designation seems 

apt if you define a syndrome as “a set of symptoms or conditions that occur 

together and suggest specific underlying factors, prognosis, or guide to treatment.” 

Like the clinical syndrome known as heart failure, there may be great utility 

in recognizing patients who meet diagnostic criteria. Although a consensus has 

not emerged, current definitions identify patients with metabolic risk factors that 

can be seen over their lifespans, from an early stage when risk factors are 

emerging until they are fully established later. Although definitions vary, some 

believe metabolic syndrome is a useful construct even for those who have developed 

diabetes. Clinicians are increasingly recognizing metabolic syndrome 

among those with CHD and stroke. Accordingly, this syndrome may be progressive 

in nature. Insulin resistance, along with weight gain and obesity, is a major 

underlying risk factor for this syndrome, but as will be seen, most definitions do 

not consider metabolic syndrome strictly synonymous with insulin resistance. 

Moreover, although metabolic syndrome predicts CHD, it is not a replacement 

for Framingham risk scoring which, according to the ATP III panel, is required 

for setting LDL-c goals to determine intensity of treatment. 4 In clinical practice, 

recognition of metabolic syndrome is a useful tool for helping both patients and 

clinical care teams understand the adverse prognosis for both diabetes and CHD 

and direct therapeutic interventions. Furthermore, those with metabolic syndrome 

face increased risks for CHD that exceed the sum of the risk factors. 8 This should 

not be surprising given the systemic factors such as inflammation and hypercoagulability 

that accompany metabolic syndrome and its attendant visceral obesity. 

3 Most important, several well controlled clinical trials have established that 

in individuals at high risk for progression to diabetes (and most likely with 

metabolic syndrome), a therapeutic lifestyle regimen directed at diet, regular 

exercise, and modest weight loss reduces the progression to type 2 diabetes by 

almost 60%. 9,10

The Metabolic Syndrome Defined 5 

Next we will consider various published definitions of metabolic syndrome 

in the hope that it will be possible to gain perspective on the merits and potential 

shortcomings of each definition. 

METABOLIC SYNDROME DEFINITIONS 

WORLD HEALTH ORGANIZATION (WHO) DEFINITION 

In 1998, the WHO proposed a set of criteria 11 to define metabolic syndrome. Its 

definition required the presence of insulin resistance as a component of the 

diagnosis. Insulin resistance was defined as the diagnosis of type 2 diabetes 

mellitus, impaired fasting glucose, impaired glucose tolerance or, for those with 

normal fasting glucose levels (30 kg/m 2 , and urinary albumin excretion rate 

>20 μg/min (Table 1.1). 

ATP III INITIAL REPORT 

In 2001, the National Cholesterol Education Program (NCEP) introduced definitions 

of metabolic syndrome, a constellation of major risk factors, life-habit risk 

factors, and emerging risk factors for CHD in its guidelines. The ATP III Expert 

Panel 12 recognized metabolic syndrome as a secondary target of risk reduction 

therapy, after the primary target of LDL cholesterol was attained. Its definition 

of metabolic syndrome included abdominal obesity, triglyceride levels, HDL 

cholesterol, blood pressure, and fasting glucose. The diagnosis of metabolic 

syndrome was made when three or more of the risk determinants were present 

(Table 1.2). 

AMERICAN ASSOCIATION OF CLINICAL ENDOCRINOLOGISTS GUIDELINES 

The guidelines of the American Association of Clinical Endocrinologists 13 appear 

to be a hybrid between the ATP III and WHO definitions of metabolic syndrome, 

but no defined number of risk factors is specified. Diagnosis is left to clinical 

judgment. The criteria include obesity, elevated triglycerides, low HDL cholesterol, 

elevated blood pressure, elevated fasting glucose and other risk factors, 

including polycystic ovary syndrome, sedentary lifestyle, advancing age, belonging 

to an ethnic group at high risk for type 2 diabetes or cardiovascular disease, 

family history of type 2 diabetes, hypertension, and cardiovascular disease 

(Table 1.3).


TABLE 1.1 

WHO Diagnostic Criteria for Metabolic Syndrome 

Component Criteria Comments 

Insulin resistance as identified 

by type 2 diabetes, impaired 

fasting glucose, or impaired 

glucose tolerance 

Any two of the following: 

body mass index; waist:hip ratio >30 kg/m 2 ; >0.9 in men or 

>0.85 in women 

For those with normal fasting 

glucose levels (88 cm (>35 inches) 

Triglycerides ≥150 mg/dL 

High-density lipoprotein cholesterol Men:


TABLE 1.3 

American Association of Clinical Endocrinologists Diagnostic Criteria 

for Insulin Resistance Syndrome 

Risk Factor Defining Level 

Elevated triglycerides >150 mg/dL 

Low HDL Men: 140 mg/dL 

Fasting glucose 110 to 125 mg/dL 

Other risk factors Family history of type 2 diabetes, hypertension, or 

cardiovascular disease; history of CVD, HTN, NAFLD, 

aeonthosis nigricans, history of glucose intolerance or 

gestational dialoetes mellitus, BMI >25 kg/m2, polycystic 

ovary syndrome; sedentary lifestyle; >40 age, membership 

in ethnic group having high risk for type 2 diabetes mellitus 

or cardiovascular disease 

Source: From Einhorn, D. et al., Endocr Pract 9, 237, 2003. 

EUROPEAN GROUP FOR STUDY OF INSULIN RESISTANCE SYNDROME 

In 1999, the European Group for Study of Insulin Resistance (EGIR) proposed 

a modification of the WHO definition. 14 This group used the term insulin resistance 

syndrome rather than metabolic syndrome. They likewise assumed that 

insulin resistance is the major cause, and required evidence of it for diagnosis. 

EGIR defines insulin resistance syndrome as the presence of insulin resistance 

or fasting hyperinsulinemia (highest 25%) and two of the following: hyperglycemia, 

hypertension, dyslipidemia, and central obesity. All these criteria must be 

measured before it is possible to evaluate the presence of the syndrome. Hyperglycemia 

should be defined at fasting values to provide reproducible criteria 

(Table 1.4). 

REVISED ATP III GUIDELINES 

In an updated version of the original ATP III guidelines, 15 the threshold for 

impaired fasting glucose (IFG) was reduced from ≥110 to ≥100 mg/dL. This 

adjustment corresponded to the recently modified American Diabetes Association 

(ADA) criteria for IFG. The rest of the criteria were essentially the same although 

several points were clarified. A diagnosis of the metabolic syndrome required 

three of the following diagnoses: abdominal obesity (as waist circumference), 

elevated triglycerides, reduced HDL cholesterol, hypertension, and elevated fasting 

glucose. As noted in ATP III, some people will manifest features of insulin 

resistance and metabolic syndrome with only moderate increases in waist


TABLE 1.4 

European Group for Study of Insulin Resistance Syndrome Criteria 


Defined by presence of insulin resistance 

or fasting hyperinsulinemia (highest 

25%) AND two of the following:* 

Hyperglycemia Fasting plasma glucose ≥6.1 mmol/L, but nondiabetic 

Hypertension Systolic/diastolic blood pressure ≥140/90 mm Hg or 

treated for hypertension 

Dyslipidemia Triglycerides >2.0 mmol/L or HDL-cholesterol 3 mg/L, microalbuminuria, 

impaired glucose tolerance, and elevated total apolipoprotein B. 

Some populations are predisposed to insulin resistance, metabolic syndrome, 

and type 2 diabetes mellitus, with only moderate increases in waist circumference 

(i.e., populations from South Asia, China, Japan, and other Asian countries). None 

of these phenotypic features or ethnic differences was included in the ATP III 

diagnostic criteria, but if individuals with such characteristics have only moderate 

elevations of waist circumference plus at least two ATP III metabolic syndrome 

features, the writing group suggests that consideration should be given to managing 

them in the same way people with three ATP III risk factors are managed 

(Table 1.5). 

INTERNATIONAL DIABETES FEDERATION (IDF) DEFINITION 

The IDF clinical definition 16 requires the presence of abdominal obesity for 

diagnosis. When abdominal obesity is present, two additional factors originally 

listed in the ATP III definition are sufficient for diagnosis: raised concentration 

of triglycerides, reduced concentration of HDL cholesterol, hypertension, and 

raised fasting plasma glucose concentration or previously diagnosed type 2 diabetes. 

IDF recognized and emphasized ethnic differences in the correlation of 

abdominal obesity and other metabolic syndrome risk factors. For this reason, 

criteria of abdominal obesity were specified by nationality or ethnicity based on


TABLE 1.5 

Revised ATP III Criteria 

Risk Factor 

(Any Three for Diagnosis) Defining Level 

Elevated waist circumference* Men: ≥102 cm (≥40 inches) 

Women: ≥88 cm (≥35 inches) 

Elevated triglycerides >150 mg/dL (1.7 mmol/L) or on drug treatment for elevated TG 

Reduced HDL cholesterol Men:


TABLE 1.6 

International Diabetes Federation Criteria for Metabolic Syndrome 


Abdominal obesity* AND two 

additional factors: 

Triglycerides ≥150 mg/dL or specific treatment for this abnormality 

Reduced concentration of HDL


lipids, diabetes, smoking, hypertension, metabolic and lifestyle factors such as 

abdominal obesity, psychosocial factors, levels of consumption of fruits and 

vegetables, alcohol intake, and regular physical activity. 

Interestingly, these investigators created a study definition of metabolic 

syndrome by using self-reported diabetes and hypertension as surrogates for 

measured fasting blood sugar and blood pressure, measured apo B and apo A1 

as surrogates for fasting triglycerides and HDL-c, and measured waist:hip ratio 

instead of waist circumference. Not surprisingly they found that their dichotomous 

metabolic syndrome variables were not as good as predictors of acute MI 

as the nine variables listed above that predicted over 90% of cases of acute MI. 

This has been a criticism of the metabolic syndrome, but in fairness, ATP III 

always emphasized that patients (especially those having two or more risk factors) 

need Framingham risk scoring with its continuous variables to calculate global 

risk and suggested attention to metabolic variables after LDL-c goals had been 

realized. For example, the ATP III algorithm notes that if triglycerides are 200 

mg/dL or more despite attainment of the LDL-c goal, then treatment to achieve 

non-HDL-c goals (some may prefer apo B) is recommended. 

It is hoped that with further investigation, consensus will eventually be 

reached on a single definition for metabolic syndrome. 

Table 1.8 summarizes unique features, drawbacks, and comments regarding 

the definitions discussed in this chapter. Endocrinologists and investigators may 

be drawn to definitions that focus on insulin resistance. Clinicians may prefer the 

easily identified clinical features of ATP III that improve with lifestyle changes 

and the resultant modest degrees of weight loss. Further studies and discussion 

hopefully will clarify the final form of the metabolic syndrome definition. Until 

then, regardless of the criteria utilized, a systematic identification of metabolic 

variables leads invariably to an appropriate strong focus on the need for improved 

diet, regular physical activity, and weight reduction that most would agree is an 

important first step in the comprehensive approach to reducing the burden of 

diabetes and CHD worldwide.


TABLE 1.8 

Features, Drawbacks, and Comments on Various Definitions of Metabolic 

Syndrome 

Defining 

Organization Unique Features Drawbacks Comments 

WHO Requires insulin resistance; 

BP requirements higher than 

ATP III; HDL cholesterol 

requirements lower; uses 

BMI instead of waist 

circumference; 

microalbuminuria is 

criterion 

2001 ATP III 

and update 

Uses abdominal obesity 

rather than BMI as criterion; 

simple criteria for diagnosis; 

update clarified several 

diagnosis issues 

AACE Uses BMI rather than 

abdominal obesity as 

criterion 

EGIR Requires insulin resistance 

for diagnosis; excludes 

patients with type 2 diabetes 

mellitus; BP requirements 

higher than ATP III; uses 

abdominal obesity rather 

than BMI as criterion; 

simple criteria for diagnosis 

IDF Requires abdominal obesity 

for diagnosis; simple criteria 

(same as ATP III) for 

diagnosis 

Requires glucose 

testing that may 

require a separate 

office or hospital 

visit 

No measure of 

insulin resistance; 

this would detract 

from ease of use of 

these criteria 

Reproducibility; no 

defined number of 

risk factors 

specified 

Insulin levels not 

reliable in clinical 

practice setting 

Physician must have 

data on waist 

circumference for 

ethnic subgroups 

Demonstrating insulin 

resistance in a patient without 

type 2 diabetes usually 

involves oral glucose 

tolerance testing or 

hyperinsulinemic/euglycemic 

clamp testing; considered 

costly or inconvenient in 

many clinical practices 

Weight loss achieved by better 

diet and regular exercise has 

potential to improve all 

components of the metabolic 

syndrome definition 

Diagnosis left to clinical 

judgment; this makes 

estimates of prognosis from 

clinical studies difficult 

Unlike some definitions, it 

excludes type 2 diabetes 

mellitus; some would argue 

that therapeutic implications 

of metabolic syndrome may 

be valuable in diabetics with 

increased waist 

circumferences 

Uses a recognizable and easily 

measurable clinical feature 

(increased waist 

circumference) to initiate 

consideration of diagnosis


REFERENCES 

1. Janssen, I. et al. Health Behaviour in School-Aged Children Obesity Working 

Group: comparison of overweight and obesity prevalence in school-aged youth 

from 34 countries and their relationships with physical activity and dietary patterns. 

Obes Rev 6, 123, 2005. 

2. Flegal, K.M. et al. Prevalence and trends in obesity among U.S. adults, 1999–2000. 

JAMA 288, 1723, 2002. 

3. Eckel, R., Grundy, S., and Zimmet, P. The metabolic syndrome. Lancet 365, 1415, 

2005. 

4. Grundy, S.M. Metabolic syndrome scientific statement by the American Heart 

Association and the National Heart, Lung, and Blood Institute. Arterioscler 

Thromb Vasc Biol 25, 2243, 2005. 

5. Vague, J. La differenciation sexuelle, facteur determinant des formes de l’obesite. 

Presse Med 30, 339, 1947. 

6. Yusuf, S. et al. INTERHEART Study Investigators: obesity and the risk of myocardial 

infarction in 27,000 participants from 52 countries: a case-control study. 

Lancet 366, 1640, 2005. 

7. Wilson, P.F.F., Kannel, W.B., Silbershatz, H., and D’Agostino, R.B. Clustering of 

metabolic risk factors and coronary heart disease. Arch Int Med 159, 1104, 1999. 

8. Grundy, S. Metabolic syndrome: connecting and reconciling cardiovascular and 

diabetes worlds. JACC 47, 1093, 2006. 

9. Diabetes Prevention Program Research Group. Reduction in the incidence of type 

2 diabetes with lifestyle intervention or metformin. New Engl J Med 346, 393, 

2002. 

10. Tuomilehto, J. et al. Finnish Diabetes Prevention Study Group: prevention of type 

2 diabetes mellitus by changes in lifestyle among subjects with impaired glucose 

tolerance. New Engl J Med 344, 1343, 2001. 

11. Alberti, K.G. and Zimmet, P.Z. Definition, diagnosis and classification of diabetes 

mellitus and its complications. Part 1. Diagnosis and classification of diabetes 

mellitus provisional report of a WHO consultation. Diabet Med 15, 539, 1998. 

12. Adult Treatment Panel (ATP) III. Executive summary of third report of the 

National Cholesterol Education Program (NCEP) Expert Panel on Detection, 

Evaluation, and Treatment of High Blood Cholesterol in Adults. JAMA 285, 2486, 

2001. 

13. Einhorn, D. et al. American College of Endocrinology position statement on the 

insulin resistance syndrome. Endocr Pract 9, 237, 2003. 

14. Balkau, B. and Charles, M.A. Comment on the provisional report from the WHO 

consultations, European Group for the Study of Insulin Resistance (EGIR). Diabet 

Med 16, 442, 1999. 

15. Grundy, S.M. et al. Diagnosis and management of the metabolic syndrome: an 

American Heart Association/National Heart, Lung, and Blood Institute scientific 

statement. Circulation 112, 2735, 2005. 

16. Alberti, K.B., Zimmet, P., and Shaw, J. The metabolic syndrome: a new worldwide 

definition. Lancet 366, 1059, 2005. 

17. Kahn, R., Buse, J., Ferrannini, E., and Stern, M. The metabolic syndrome: time 

for a critical appraisal. A joint statement from the American Diabetes Association 

and the European Association for the Study of Diabetes. Diabet Care 28, 2289, 

2005.


18. Yusuf, S. et al. Effect of potentially modifiable risk factors associated with myocardial 

infarction in 52 countries (the INTERHEART study): case-control study. 

Lancet 364, 937, 2004.

2 

CONTENTS 

The Metabolic 

Syndrome: The Question 

of Balance between the 

Pro-Inflammatory Effect 

of Macronutrients and 

the Anti-Inflammatory 

Effect of Insulin 

Paresh Dandona, Ajay Chaudhuri, 

Priya Mohanty, and Husam Ghanim 

Introduction .........................................................................................................15 

Insulin Resistance Syndrome: Metabolic Perspective........................................16 

Novel Non-Metabolic Actions of Insulin ...........................................................17 

Obesity and Inflammation...................................................................................20 

Insulin Resistance: Inflammatory Hypothesis ....................................................22 

Macronutrients and Origin of Inflammation ......................................................22 

Conclusion: Inflammation Hypothesis of Metabolic Syndrome........................25 

References ...........................................................................................................26 

INTRODUCTION 

The common occurrence of the combination of obesity, insulin resistance, hypertension, 

hypertriglyceridemia, low HDL cholesterol, and hyperinsulinemia was 

first described by Reaven as insulin resistance syndrome or metabolic syndrome. 

The syndrome was recognized as a pro-atherogenic risk causing coronary heart 

disease (CHD). More recently, other features like elevated plasma PAI-1 and CRP 

15


have been added to the combination. In view of recent data demonstrating that 

insulin exerts an anti-inflammatory effect while macronutrients exert pro-inflammatory 

effects, we are in a better position to explain why an insulin-resistant 

state such as metabolic syndrome is pro-inflammatory and also explain how it 

develops. This focused review discusses the relevance of these recent observations, 

puts into perspective the pathogenesis of various features of metabolic 

syndrome, and also predicts some features that may be incorporated into it in the 

future. 

INSULIN RESISTANCE SYNDROME: METABOLIC PERSPECTIVE 

Reaven’s original description of the metabolic syndrome consisted of obesity, 

insulin resistance, hypertension, impaired glucose tolerance or diabetes, hyperinsulinemia, 

and dyslipidemia characterized by elevated triglyceride and low 

HDL concentrations. 1 All these features serve as risk factors for atherosclerosis 

and thus metabolic syndrome constitutes a significant risk for coronary heart 

disease 2–5 (Table 2.1). The features of obesity or overweight and insulin resistance 

also provide significant risks for developing type 2 diabetes. 5,6 The risks for CHD 

and diabetes with metabolic syndrome are greater than those for simple obesity 

alone without insulin resistance and therefore the understanding of the pathogenesis 

and, through it, a rational approach to its therapy are of prime importance. 

TABLE 2.1 

Classic Biological Effects of Insulin and Classic Metabolic Syndrome 

Based on Resistance to Metabolic Effects of Insulin 

Nutrients Normal Insulin Action Insulin-Resistant State 

Carbohydrates Hepatic glucose production 

Glucose utilization 

Glycogenesis 

Lipids Lipolysis 

FFA and glycerol 

Lipogenesis 

HDL 

Triglycerides 

Proteins Gluconeogenesis 

Amino acids 

Protein synthesis 

Purines Uric acid clearance 

Uric acid formation 

Hyperglycemia 

Hyperinsulinemia 

Source: From Dandona, P. et al., Circulation 111, 1448, 2005. 

Lipolysis 

FFA and glycerol 

Hepatic triglyceride and apo B synthesis 

Hypertriglyceridemia 

HDL 

Small dense LDL 

Glyconeogenesis 

Protein catabolism 

Protein synthesis 

Hyperuricemia

The Metabolic Syndrome 17 

The original conceptualization of this syndrome was based on resistance to 

the metabolic actions of insulin. Thus, hyperinsulinemia, glucose intolerance, 

type 2 diabetes, hypertriglyceridemia, and low HDL concentrations could be 

accounted for by resistance to the actions of insulin on carbohydrate and lipid 

metabolism. While the features described above explain the atherogenesis to some 

extent, Reaven maintained that hyperinsulinemia contributed to atherogenicity 

and thus insulin is atherogenic, leading to CHD and cerebrovascular disease 

associated with this syndrome. However, as our understanding of the action of 

insulin evolves to comprehensively include recent discoveries, 7 we can better see 

that insulin resistance is the basis of most if not all the features of this syndrome. 

Obesity probably leads to hypertension through (1) increased vascular tone 

created by a reduced bioavailability of nitric oxide (NO) due to increased oxidative 

stress, 8 (2) increased asymmetric dimethyl arginine (ADMA) concentrations, 9 

(3) increased sympathetic tone, 10 and (4) increased expression of angiotensinogen 

by adipose tissue leading to an activation of the renin–angiotensin system. 11 The 

last factor requires further critical investigation. 

Metabolic syndrome is characterized by low HDL in association with an 

elevated triglyceride concentration. This is believed to be due to an increased 

triglyceride load in HDL particles that are acted upon by hepatic lipase that 

hydrolyzes the triglyceride. The loss of the triglyceride results in a small HDL 

particle filtered by the kidney, resulting in decreases in apolipoprotein A (apo A) 

and HDL concentrations. Apart from an increase in the loss of apo A, data 

demonstrate that insulin may promote apo A gene transcription. 12 Therefore, 

insulin-resistant states may be associated with diminished apo A biosynthesis. 13 

An increase in plasma free fatty acid (FFA) concentrations plays a key role 

in the pathogenesis of insulin resistance through specific actions that block insulin 

signal transduction. Increases of plasma FFA concentrations in normal subjects 

to levels comparable to those in the obese also resulted in the induction of 

oxidative stress, inflammation, and subnormal vascular reactivity along with 

insulin resistance. 14 Because resistance to insulin also results in the relative nonsuppression 

of adipocyte hormone-sensitive lipase, there is further enhancement 

of lipolysis and an increase in FFA concentration, leading to a vicious cycle of 

lipolysis, increased FFA, insulin resistance, and inflammation. 

Several new features have been added to the syndrome over time. These 

include elevated plasminogen activator inhibitor-1 (PAI-1) concentrations and 

elevated C-reactive protein (CRP) concentrations. These features were added on 

the basis that they were found frequently in association with metabolic syndrome 

and no rational explanation indicated why they actually occurred. These features 

are probably related to both insulin resistance and obesity. The relationship of 

inflammation to obesity and insulin resistance still needs to be explained. 15 

NOVEL NON-METABOLIC ACTIONS OF INSULIN 

The nonmetabolic actions of insulin are readily explained by the recent observations 

that insulin is an anti-inflammatory hormone and that macronutrient intake is


pro-inflammatory. Insulin has been shown to suppress several pro-inflammatory 

transcription factors such as nuclear factor kappa-B (NFκB), early growth response- 

1 (Egr-1), activator protein-1 (AP-1), and the corresponding genes they regulate 

that mediate inflammation. 16,17 An impairment of the action of insulin due to insulin 

resistance would thus result in the activation of these pro-inflammatory transcription 

factors and an increase in the expression of the corresponding genes. 

Insulin has been shown to suppress NFκB binding activity, reactive oxygen 

species (ROS) generation, p47 phox expression, increase inhibitor kappa-B (IκB) 

expression in mononuclear cells (MNCs), and suppress plasma concentrations of 

intracellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 

(MCP-1). 16 In addition, insulin suppresses AP-1 and Egr-1, two pro-inflammatory 

transcription factors and their respective genes, matrix metalloproteinase- 

9 (MMP-9), tissue factor (TF), and PAI-1. 17–19 Thus, insulin exerts comprehensive 

anti-inflammatory effects and also has anti-oxidant effects as reflected in the 

suppression of ROS generation and p47 phox expression (Figure 2.1). 16,20 

Two further pieces of evidence demonstrating the anti-inflammatory action of 

insulin have emerged recently. First, the treatment of type 2 diabetes with insulin for 

2 weeks caused reductions in CRP and MCP-1. 21 Second, the treatment of severe 

hyperglycemia associated with marked increases in inflammatory mediators with 

insulin resulted in rapid marked decreases in the concentrations of inflammatory 

mediators. 22 Most recently, in a rat model in which inflammation was induced with 

endotoxin, insulin suppressed the concentration of inflammatory mediators including 

Platelet Inhibition 

↑ NO release in platelets 

↑ e-AMP 

Novel Biological 

Action of Insulin 

Vasodilation Cardio-protective 

↑ NO release 

↑ eNOS expression 

Animals, human 

Vascular (other) actions 

Anti-apoptotic 

Heart, other issues 

Anti-oxidant Anti-inflammatory Anti-thrombotic Profibrinolytic Anti-atherosclerotic 

↓ ROS generation ↓ NFκB, ↑ lκB 

↓ MCP 

↓ ICAM-1 

↓ CRP 

↓ TF ↓ PAI-1 Apo E null mouse 

IRS-1 null mouse 

IRS-2 null mouse 

FIGURE 2.1 Novel biological effects of insulin targeted at endothelial cells, platelets, 

and leucocytes resulting in vasodilation, anti-aggregatory effects on platelets, anti-inflammatory 

effects, and other related effects. (Source: From Dandona, P. et al., Circulation 

111, 1448, 2005.)


interleukin-1 beta (IL-1β), IL-6, macrophage inhibitory factor (MIF), and tumor 

necrosis factor alpha (TNFα). 23 Insulin also suppressed the expression of pro-inflammatory 

transcription factor CEBP and cytokines in the livers of the experimental 

animals. Similar reductions in inflammatory mediators were observed in rats with 

thermal injuries treated with insulin. 24 Finally, insulin has been shown to suppress 

the increases in cytokine concentrations in pigs challenged with endotoxin. 23 

The anti-inflammatory, anti-oxidant (ROS-suppressive), anti-thrombotic, and 

pro-fibrinolytic effects of insulin have recently been shown to occur in patients with 

acute myocardial infarctions when they were treated with low dose infusions of 

insulin independently of decreases in glucose concentrations. These patients demonstrated 

impressive 40% reductions in plasma CRP and serum amyloid A (SAA) 

concentrations at 24 hours. This reduction was maintained at 48 hours of insulin 

infusion. 25 

These anti-inflammatory effects of insulin have also been shown in patients 

undergoing coronary artery bypass grafts in association with extracorporeal circulation. 

26 The increase in plasma CRP concentration occurring within 16 hours 

of surgery is 30 times greater than that in patients with ST-Elevation Myocardial 

Infarction (STEMI). 26 The reduction in the magnitude of increase in CRP and 

SAA is 40% — very similar to that observed following insulin infusion in patients 

with STEMI. 26 

Another novel anti-apoptotic effect of insulin has recently been described. In 

experimental acute myocardial infarction in the rat heart, the addition of insulin 

to the reperfusion fluid led to a 50% reduction in infarct size. 27 More recently, a 

similar cardio-protective effect of insulin was shown in human acute myocardial 

infarction when insulin at a low dose was infused with a thrombolytic agent and 

heparin. 20 Conversely, insulin-resistant states of obesity and type 2 diabetes have 

been shown to be associated with larger infarcts than those observed in nondiabetic 

subjects. Further work is required to establish this feature as an integral 

component of metabolic syndrome. 

It should also be mentioned that insulin administration suppresses atherogenesis 

in apolipoprotein E null mice. 28 Conversely, interference with insulin signal 

transduction, as in IRS-2 null mice, resulted in atherosclerosis. 29 The IRS-1 null 

mouse also has a tendency toward atherosclerosis. It is relevant that a mutation 

of IRS-1 (arginine at 792) leads to abnormal vascular reactivity, a decrease in 

eNOS expression in endothelial cells, and an increased incidence of coronary 

heart disease. 30 It is interesting that the pro-atherogenic effects of insulin proposed 

primarily on the basis of in vitro studies are being challenged by evidence 

generated in the past 6 years. 7 This debate has been further fueled by two recent 

articles showing that knocking out the insulin receptor in myelogenic cells that 

are precursors of MNCs (cells that play a crucial role in the pathogenesis of 

atherosclerosis) is anti-atherogenic in the background of LDL receptor deficiency 

and pro-atherogenic in apo E-deficient animals. 31,32 

Consistent with the anti-inflammatory effects of insulin, insulin sensitizers 

of the thiazolidinediones class (troglitazone 33,34 and rosiglitazone 35 ) have been 

shown to exert anti-inflammatory effects in addition to their glucose lowering


effects in patients with diabetes. Troglitazone has been shown to suppress the 

development of diabetes in patients at high risk of developing this condition. 36 

Trials are under way to determine whether rosiglitazone and pioglitazone prevent 

both type 2 diabetes and atherosclerotic complications. Positive results from those 

trials would support the concept that inflammatory mechanisms underlie the 

pathogenesis both of insulin resistance and atherosclerosis. It is of interest in this 

regard that metformin causes reductions in plasma concentrations of MIF in obese 

subjects. 37 Obese individuals have elevated plasma concentrations of this cytokine 

and increases in the expression of this cytokine in MNCs. 37 While evidence 

indicates that TZDs exert direct anti-inflammatory effects on macrophages in 

vitro, it is possible that their effects in vivo may arise through insulin sensitization. 

OBESITY AND INFLAMMATION 

The above data explain why an insulin-resistant state may be pro-inflammatory. 

They do not, however, explain the origin of insulin resistance. Mutations of the 

genes involved in insulin signal transduction provide one approach to the study 

of this issue in humans and in mice with specific gene knockouts. Such lesions 

are of interest but are too infrequent to provide a basis for the understanding of 

the pathogenesis of insulin resistance at large in humans. Thus, some recent 

observations on the interference of insulin signal transduction by inflammatory 

mechanisms are of great interest because obesity is a pro-inflammatory state 

(Figure 2.2). 

Even if we accept that inflammatory mechanisms are involved in the pathogenesis 

of interference with insulin signal transduction and of insulin resistance 

itself, how does inflammation arise? Over the past decade, obesity has been 

associated with inflammation. This association was first proposed in a landmark 

paper by Hotamisligil et al. in which TNFα was shown to be constitutively 

expressed by adipose tissue, to be hyper-expressed in obesity, and to mediate 

insulin resistance in the major animal models of obesity. 38 This seminal paper 

also demonstrated that the neutralization of TNFα with soluble TNFα receptors 

resulted in the restoration of insulin sensitivity. Thus, the TNFα pro-inflammatory 

cytokine was the mediator of insulin resistance. Although the infusion of soluble 

TNFα receptors in humans has not reproduced the results observed in mice, 39 

Hotamisligil’s paper laid the foundation of the concept that inflammatory mechanisms 

may have a role to play in the pathogenesis of insulin resistance. 

More data have now accumulated to reinforce the concept that obesity is an 

inflammatory state in humans. Increased plasma concentrations of TNFα, IL-6, 

CRP, MIF, and other inflammatory mediators were demonstrated in obese subjects. 

37,40–44 Adipose tissue has been shown to express most of these pro-inflammatory 

mediators. It has also been shown that macrophages residing in adipose 

tissue may also be sources of pro-inflammatory factors and may also modulate 

the secretory activities of adipocytes. 45


Platelet 

Hyperaggregability 

↓ NO 

↓ PGl 2 

↑ ROS generation 

↑ Oxidative stress 

↑ NADPH oxidase 

↑ Mitochondrial superoxide 

Tonic vasconstriction 

-abnormal vascular reactivity 

-↓ vascular flow reserve 

Chronic pro-inflammatory state 

↑ NFκB 

↓ IκB 

↑ MIF 

↑ CRP 

↑ TNFα 

↓ NO bioavailability 

↑ ADMA 

Pro-thrombotic state 

↓ Cardio-protection 

Novel features of metabolic 

syndrome on the basis of the 

vascular and other effects 

of insulin 

Larger infarcts 

↑ Tendency to CHF 

Pro-apoptotic state 

↑ Infarct size 

Atherosclerosis 

CHD 

Stroke 

Anti-fibrinolytic state 

FIGURE 2.2 Extension of metabolic syndrome based on novel actions of insulin. (Source: 

From Dandona, P. et al., Circulation 111, 1448, 2005.) 

Tissue macrophages are derived from monocytes in blood. Recently, the 

mononuclear cells of obese patients, of which monocytes constitute a fraction, 

have also been shown to be in an inflammatory state, expressing increased 

amounts of pro-inflammatory cytokines and related factors. 46 In addition, these 

cells have been shown to have significantly increased binding of NFκB, the key 

pro-inflammatory transcription factor, and an increase in the intranuclear expression 

of p65 (Rel A), the major protein component of NFκB. These cells also 

express diminished amounts of IκBβ, the inhibitor of NFκB. Evidence of inflammation 

clearly exists in various cells and in plasma in obesity. 

In addition to TNFα and IL-6, the major adipocyte cytokines, three other 

important proteins, leptin, adiponectin, and resistin, need mention. While leptin is 

known for its function as a satiety signal that inhibits feeding, it has additional roles 

as a regulator of sexual function and as an immune modulator. It is also proinflammatory 

and induces platelet aggregation. 47–49 Thus, its elevated concentrations 

may contribute to the pro-inflammatory state of obesity and to atherogenesis in the 

long term. On the other hand, adiponectin, secreted in abundance by adipocytes in 

normal subjects, is anti-inflammatory and thus potentially anti-atherogenic. In contrast 

to leptin, its concentration falls with weight gain and in obesity. 50,51 It has been 

suggested that a low adiponectin concentration may be a marker for atherosclerosis 

and coronary heart disease. 52 Furthermore, in several experimental models, it has 

been shown to be protective to the arterial endothelium. 

↑ TF 

↑ PAl-1


Resistin, discovered as a gene suppressed by rosiglitazone in mouse adipose 

tissue, earned its name because it induced insulin resistance. 53 Thiazolidenediones 

(TZDs) suppress resistin concentrations in humans while inducing an increase in 

adiponectin concentration consistent with the anti-inflammatory effects of these 

drugs. Furthermore, it has been shown that the increase in adiponectin and the 

decrease in resistin occur early after rosiglitazone treatment along with manifestations 

of other anti-inflammatory effects prior to any changes in plasma concentrations 

of insulin, glucose, or FFAs. Thus, these early anti-inflammatory effects 

are independent of the metabolic effects of TZDs. 54 

INSULIN RESISTANCE: INFLAMMATORY HYPOTHESIS 

Which aspect of the inflammatory state results in insulin resistance? The first of 

these potential mechanisms was described by Hotamisligil et al., who demonstrated 

that TNFα induced serine phosphorylation of IRS-1 which in turn caused 

the serine phosphorylation of the insulin receptor. This prevented the normal 

tyrosine phosphorylation of the insulin receptor and thus interfered with insulin 

signal transduction. 55 IL-6 and TNFα have recently been shown to induce suppressor 

of cytokine signaling-3 (SOCS-3). 56,57 This protein was hitherto thought 

to interfere with cytokine signal transduction but is now also known to interfere 

with tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 

(IRS-1) and cause ubiquitination and proteosomal degradation of IRS- 

1. 58 This in turn reduces the activation of Akt (protein kinase B) which normally 

causes the translocation of the Glut-4 insulin-responsive glucose transporter to 

the plasma membrane. It also induces the phosphorylation of the nitric oxide 

synthase (NOS) enzyme and its activation to generate NO. 59 

A newly described protein known as TRB3 has also been shown to interfere 

with the activation of Akt and thus to interfere with insulin action. 60 However, the 

association of TRB3 with inflammatory mechanisms has not been demonstrated. 

Recent data indicate that Akt2, a key protein involved in insulin signal 

transduction that mediates the phosphorylation and activation of e-NOS and NO 

secretion, also prevents the mobilization of Rac-1 to cell membranes, thus preventing 

superoxide generation. Superoxide generation is dependent upon the 

translocation of essential elements of NADPH oxidase (e.g., p47 phox ) from the 

cytosol to the membrane. This is mediated by Rac. 61 In the absence of Akt2, there 

will be an increase in the translocation of Rac-1 to the membrane, greater formation 

of NADPH oxidase complex, increased superoxide generation, and oxidative 

stress. It has been shown that Akt2 null mice develop insulin resistance 

and mild hyperglycemia in association with hyperinsulinemia. 62 

MACRONUTRIENTS AND ORIGIN OF INFLAMMATION 

If indeed obesity is a pro-inflammatory state and inflammatory mechanisms interfere 

with insulin signal transduction, what is the origin of this pro-inflammatory 

state? The answer comes mainly from recent observations demonstrating that


macronutrient intake may induce oxidative stress and inflammatory responses. 

Thus a 75-g glucose challenge has been shown to induce an increase in superoxide 

generation by leucocytes by 140% over the basal in addition to increasing p47 phox 

expression, a subunit of NADPH oxidase, the enzyme that converts molecular O 2 

to superoxide radical. 63 

Equicaloric amounts of cream (fat) intake result in similar amounts of oxidative 

stress. 64 Glucose intake also results in comprehensive inflammation as 

reflected in an increase in intranuclear NFκB binding, a decrease in IκB expression, 

and an increase in IKKα and IKKβ, the two kinases that phosphorylate 

IκBα and IκBβ and result in their ubiquitination and proteosomal degradation. 65 

Glucose intake also causes increases in two other pro-inflammatory transcription 

factors: AP-1 and Egr-1. 66 AP-1 regulates the transcription of matrix metalloproteinases 

while Egr-1 modulates the transcription of tissue factor (TF) and PAI-1. 

Thus, glucose intake increases the expression of matrix metalloproteinases 2 and 

9 as well as expression of TF and PAI-1. 

A mixed meal from a fast food chain was also shown to induce the activation 

of NFκB, a reduction in IκBα, an increase in IKKα and IKKβ, and an increase 

in superoxide radical generation by MNCs. 67 It is also of interest that an intravenous 

infusion of triglyceride with heparin in normal subjects with elevations 

of FFA concentrations to levels comparable to those found in obese subjects 

resulted in inflammatory responses. 14 

All genes that are stimulated by acute nutritional intake have also been shown 

to be activated in the basal states of obese subjects such that the concentrations 

of these gene products were elevated. Consistent with this, reductions in macronutrient 

intake in obese subjects (1000 kcal daily for 4 weeks) were shown to 

reduce both oxidative stress and inflammatory mediators. 8 Similarly, a 48-hour 

fast has been shown to reduce ROS generation by over 50% in normal subjects; 

the expression of p47 phox was also reduced. 68 

Clearly, macronutrient intake is a major regulator of oxidative stress. It is 

relevant that the superoxide radical generated during oxidative stress is an activator 

of at least two major pro-inflammatory transcription factors, NFκB and AP- 

1. NFκB regulates the transcriptional activities of at least 200 genes, most of 

which are pro-inflammatory. 69–72 Thus, it is not surprising that obesity is a proinflammatory 

condition. Indeed, the MNCs of obese individuals are in a proinflammatory 

state, expressing an excess of a series of pro-inflammatory genes 

in addition to demonstrating increased NFκB binding, p65 expression, and 

decreased IκBβ protein. 46 

In addition to obesity and increased macronutrient intake, genetic and other 

environmental factors may induce the activation of inflammatory mechanisms and 

the induction of oxidative stress. These factors may be relevant in those ethnic 

groups in whom metabolic syndrome has been shown to occur in the absence of 

obesity. In these groups, migration to western countries like the U.S. and U.K. 

results in increased adiposity with a sedentary lifestyle that results in a phenotype 

of the metabolic syndrome against an appropriate genetic background (Figure 2.3).


Obesity 

↑ Food Intake 

↑ ROS, ↑ p47 

(Oxidative Stress and Inflammation) 

phos 

↑ NFκB 

↑ AP-1, ↑ MMPs 

↑ Egr-1, ↑ TF, ↑ PAI-1 

Serine Phosphorylation of IRS-1 

↑ SOCS-3 

Interruption of Insulin Signal Transduction 

Insulin Resistance 

↑ Lipolysis 

↑ FFA 

Genetic Factors 

FIGURE 2.3 Pathogenesis of metabolic syndrome. (Source: From Dandona, P. et al., 

Circulation 111, 1448, 2005.) 

When considering macronutrient-induced inflammation, it can be argued that 

the foods consumed now were always consumed, so why are their pro-inflammatory 

effects suddenly becoming relevant? The reason is that the amounts of food consumed 

now are far greater; furthermore, larger portions of the average diet consist 

of fast foods and lack sufficient fiber, fruits, and vegetables. Furthermore, the 

relative contents of refined carbohydrates and saturated fats have increased. This 

combination results in the inability of endogenously secreted insulin in response 

to meal intake to suppress the inflammation generated by the meal. 

It is of interest in this regard that a 900-kcal AHA step 2 diet-based meal 

rich in fruit and fiber does not cause significant oxidative stress or inflammation 

in contrast to the effect of an isocaloric fast food meal. 73 Furthermore, orange 

juice taken in amounts equivalent to 75 g glucose (= 300 cal) does not induce 

any increase in O 2 • generation or an increase in NFB binding by MNCs. This 

may be due partly to the anti-oxidant and anti-inflammatory effects of ascorbic 

acid and flavanoids contained in orange juice. However, it has also been shown 

that fructose, which accounts for 50% of carbohydrates in orange juice, does not 

cause O 2 • generation or increased NFB binding. It is also of interest that orange 

juice induced a greater increase in insulin for a certain increase in glucose 

concentration when compared to glucose challenge. 74 This raises a novel concept 

when considering post-prandial hyperglycemia, oxidative stress, and inflammation 

following ingestion of various foods. The recent demonstration that M3 

muscarinic receptors in the β cells of the pancreatic islets enhanced the insulinogenic 

response to a given glucose load or concentration suggests such an avenue


of investigation. It is possible that certain foods like orange juice activate and/or 

increase the expression of M3 muscarinic receptors on β cells to enhance insulin 

secretion in response to glucose challenge. 75 

Both glucose and cream intake induce the expression of a series of proinflammatory 

genes in peripheral blood MNCs as reflected in the mRNA. These 

genes include TNFα, IL-6, and other related genes. Among the genes induced 

are SOCS-3, the molecule considered a potential candidate for the mediation of 

insulin resistance through serine phosphorylation, ubiquitination, and proteasomal 

degradation of IRS-1. 

The increase in superoxide radical generation also results in diminished 

bioavailability of NO since NO binds to superoxide radical to form peroxynitrate. 

76 In addition to the fact that Akt is inhibited due to insulin resistance, and 

thus NOS is also inhibited, the reduction in NO bioavailability can result in a 

marked reduction in NO action. Furthermore, TNFα suppresses the expression 

of NOS. The factors result in abnormalities in endothelium-mediated vasodilatation 

and vascular reactivity. 77 Interestingly, the abnormalities in vascular reactivity 

in the obese insulin-resistant population can be reproduced acutely by a 900-kcal 

fast food meal, just as the pro-inflammatory changes in obesity can be reproduced 

by a similar meal. 67 It is noteworthy that the plasma concentrations of asymmetric 

dimethylarginine (ADMA) are elevated in obesity and inhibit NOS activity, thus 

reducing the synthesis and secretion of NO. 78 Rosiglitazone suppresses plasma 

ADMA concentrations while improving the impaired vascular reactivity in the 

obese and those with type 2 diabetes. 79 

While the initial work on macronutrient intake with glucose, cream, and fast 

food meals showed a pro-inflammatory effect associated with oxidative stress, 

data are now emerging to demonstrate that some macronutrients may be safe and 

non-inflammatory. Thus, a 900-kcal breakfast rich in fruit and fiber does not cause 

oxidative stress or inflammation. The intake of vitamin E prior to glucose challenge 

also suppresses oxidative stress and inflammation. Similarly, alcohol 65 and 

orange juice in equicaloric amounts do not cause oxidative stress or inflammation. 

Because orange juice is rich in flavanoids and vitamin C, it is possible that the 

presence of macronutrients in food may alter or suppress oxidative stress or 

inflammation. Furthermore, data show that vitamin E administration to patients 

with insulin resistance reduced cytokine production by MNCs. 80 

Other strategies to prevent post-prandial oxidative stress and inflammation 

may involve drug therapies. One initial attempt in this area indicates that rosiglitazone 

treatment for 6 weeks (8 mg/day) led to the total suppression of oxidative 

and inflammatory stress caused by a 75-g glucose challenge in type 2 diabetes. 81 

CONCLUSION: INFLAMMATION HYPOTHESIS OF 

METABOLIC SYNDROME 

In conclusion, the pro-inflammatory state of obesity and metabolic syndrome 

originates with excessive caloric intake and is probably due to over-nutrition in


a majority of patients in the U.S. The pro-inflammatory state induces insulin 

resistance leading to clinical and biochemical manifestations of the metabolic 

syndrome. This resistance to insulin action further promotes inflammation 

through increases in lipolysis and plasma FFA concentrations on one hand and 

interference with the anti-inflammatory effect of insulin on the other. 

While these factors may be the most important ones in a majority of patients 

with metabolic syndrome, it is possible that genetic factors may also contribute 

to the inflammatory stress in metabolic syndrome. These factors may be important 

in ethnic groups such as Asian Indians who may have increased amounts of upper 

abdominal fat in spite of normal BMI values. 82 Since excessive nutritional intake 

probably accounts for the inflammation at least in obesity-associated metabolic 

syndrome, the most rational way to suppress such inflammation is through caloric 

restriction. 

The other lifestyle change that affects inflammation is exercise. Exercise 

produces a decrease in the indices of inflammation such as plasma CRP concentration. 

83 The mechanism underlying this effect is not known. However, it is 

noteworthy that a lifestyle change is a very effective way to reduce the rate of 

development of diabetes in a pre-diabetic population as shown by the diabetes 

prevention study. 84,85 Exercise and reductions in macronutrient intake both cause 

a reduction in inflammation. Several drugs have also been shown to reduce 

oxidative stress and inflammation and may therefore be used in the treatment of 

metabolic syndrome. Among them are thiazolidinediones, angiotensin II receptor 

blockers, and carvedilol, a β-blocker with some α-blocking activity. 33,35,54,86–88 

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3 

CONTENTS 

The Role of Oxidative 

Stress in Diseases 

Associated with 

Overweight and 

Obesity 

Ginger L. Milne, Ling Gao, Joshua D. Brooks, 

and Jason D. Morrow 

Introduction .........................................................................................................33 

F 2-Isoprostanes as Markers of Oxidant Stress In Vivo.......................................34 

F 2-Isoprostanes and Overweight and Obesity ....................................................35 

Therapeutic Targets for Reducing Oxidant Stress in Overweight and 

Obese Patients ...........................................................................................38 

Antioxidants...............................................................................................38 

ω-3 Polyunsaturated Fatty Acids ..............................................................39 

Conclusions .........................................................................................................41 

Acknowledgments ...............................................................................................42 

References ...........................................................................................................42 

INTRODUCTION 

The marked increase in the incidence of overweight and obese persons is recognized 

as perhaps the most serious public health issue in the United States. It is 

estimated that two-thirds of American adults are overweight and nearly 30% are 

obese. 1,2 Additionally, the incidence of overweight and obesity in children and 

adolescents is rising; in 2004, it was estimated that 16% of youth are either 

overweight or obese. 1,3 Both morbidity and mortality increase with excessive 

body weight. 4–6 

33


Multiple studies have shown that the risks of developing cardiovascular disease, 

type 2 diabetes mellitus, hypertension, dyslipidemia, gallbladder disease, 

osteoarthritis, stroke, and certain types of cancers increase with degree of overweight 

in both men and women. 2,7 Furthermore, early onset of symptoms of these 

diseases that were once only associated with adulthood are now observed in 

overweight adolescents and include high blood pressure, atherosclerosis, and type 

2 diabetes mellitus. 8 Despite the well characterized association of overweight and 

disease incidence, the mechanisms by which overweight contributes to disease 

pathology are poorly understood. Nonetheless, several reports provide evidence 

that elevated systemic oxidant stress may be an important mechanism by which 

obesity promotes the development of chronic human disease. 9–11 

This chapter will examine the measurement of F 2-isoprostanes (IsoPs), a 

class of oxidized lipids generated from the peroxidation of arachidonic acid, as 

a marker of oxidant stress in overweight, obesity, and associated diseases. It 

will also assess the impacts of select therapeutic interventions on F 2-IsoP formation 

in this population. 

F 2-ISOPROSTANES AS MARKERS OF OXIDANT STRESS IN VIVO 

F 2-IsoPs represent one class of oxidized lipids formed in vivo in humans. 12–14 

These compounds that were first identified by our laboratory in 1990 are prostaglandin 

(PG)-like molecules generated non-enzymatically from the free radicalinitiated 

peroxidation of arachidonic acid, a ubiquitous polyunsaturated fatty 

acid. 15 Figure 3.1 shows the mechanism of formation of the F 2-IsoPs. Briefly, 

after abstraction of a bis-allylic hydrogen atom, one molecule of oxygen adds to 

arachidonic acid to form a peroxyl radical. This peroxyl radical then undergoes 

5-exo cyclization and subsequent addition of another molecule of oxygen to form 

PGG 2-like compounds. 

The unstable bicyclic endoperoxide intermediates are then reduced to the F 2- 

IsoPs, isomers of the cyclooxygenase-derived product PGF 2α. Based on this mechanism 

of formation, four series of F 2-IsoP regioisomers are generated. In total, 64 

different F 2-IsoP stereoisomers are formed from the oxidation of arachidonic acid. 

In more recent years since their discovery, several methods have been developed 

to quantify the formation of F 2-IsoPs in vivo. 16 The method our laboratory 

and others found to be most sensitive, specific, and reliable employs the use of 

mass spectrometry and stable isotope dilution techniques. 17 Employing this technique, 

F 2-IsoPs have been detected in all human tissues and fluids examined, 

which is of particular importance in that it allows for an assessment of the effects 

of diseases on endogenous oxidant tone. 14 Additionally, defining levels of F 2- 

IsoPs in vivo is important because it allows for the determination of the extent 

to which various therapeutic interventions affect levels of oxidant stress. 

For human studies, the quantification of F 2-IsoPs in body fluids such as urine 

and plasma is significantly more convenient and less invasive than measuring 

these compounds in organ tissue. Based on available data, quantification of these 

compounds in either plasma or urine is representative of their endogenous

Oxidative Stress in Diseases Associated with Overweight and Obesity 35 

O O 

O 

O 

O 

O 

O 

O 

HO 

10 

HO 

COOH 

COOH 

COOH 

O 

O 

Arachidonic acid 

O 

OH 

COOH 

O 

O 

O O 

COOH COOH 

COOH 

O 

O 

FIGURE 3.1 Mechanism of F 2-isoprostane formation from the free radical-initiated peroxidation 

of arachidonic acid. 

production, and thus gives a highly precise and accurate index of in vivo oxidant 

stress. 14,16,18,19 In fact, since their initial discovery, quantification of the F 2-IsoPs 

by mass spectrometry has emerged as the “gold standard” index of in vivo oxidant 

stress status. 

Recently, this assertion was independently confirmed by a National Institutes 

of Health-sponsored program termed the Biomarkers of Oxidative Stress Study 

(BOSS) that directly compared measurements of plasma and urinary F 2-IsoPs 

with other well known but less robust biomarkers of oxidant stress, including 

malondialdehyde (MDA) and other measures of lipid peroxidation, plasma glutathione, 

plasma antioxidant levels, protein carbonyls, and 8-hydroxy-deoxyguanosine. 

20,21 

F 2-ISOPROSTANES AND OVERWEIGHT AND OBESITY 

As discussed above, measurement of plasma or urinary F 2-IsoPs allows for an 

assessment of the effects of diseases on oxidant tone in vivo. In the past decade, 

O O 

COOH O 

O 

O2 O2 O2 O2 OO 

COOH 

O 

O 

COOH 

O 

OO 

O 

COOH 

O 

OO 

O 

OO 

OOH 

COOH 

O 

O 

COOH 

O 

OOH 

O 

COOH 

O 

OOH O 

OOH 

[H] [H] [H] [H] 

COOH 

COOH 

COOH 

COOH 

COOH 

OH 

COOH 

HO 

COOH 

HO 10 

10 

HO 10 

OH 

HO 

5-series IsoP 12-series IsoP 8-series IsoP 15-series IsoP 

OH 

HO 

COOH 

COOH 

HO 

OH


quantification of F 2-IsoPs has been used to implicate a role for oxidative stress 

in the pathophysiology of a number of human conditions and diseases. Notably, 

F 2-IsoP levels were shown to be increased in neurodegenerative conditions such 

as Alzheimer’s disease, Huntington’s disease, aging, certain types of cancers, and, 

of notable importance to consequences of overweight and obesity, atherosclerotic 

cardiovascular disease. 14,22–26 

A role for oxidant stress in the development and progression of atherosclerosis 

has been hypothesized for more than two decades. 27–29 In recent years, however, 

the quantification of F 2-IsoPs has allowed investigators to explore, for the first 

time, the extent to which humans undergo enhanced oxidant stress under pathophysiological 

situations associated with the development of atherosclerotic cardiovascular 

disease. These studies have found that increased levels of plasma 

and/or urinary F 2-IsoPs are associated with most of the risk factors for atherosclerosis, 

including hypercholesterolemia, 30 diabetes mellitus, 31–33 hyperhomocysteinemia, 

34 and chronic cigarette smoking. 35–37 This suggests that certain populations 

at risk for the disease are under increased oxidant stress. 

Oxidant stress levels in the overweight and obese, a population at significant 

risk for both atherosclerosis and also for its associated risk factors in which 

oxidant stress is increased, had not been studied until recently. Three interesting 

studies, however, have independently reported that the overweight and obese 

population is under increased oxidant stress. 

In the first of these studies, Block and colleagues at the University of California 

at Berkeley and Kaiser Permanente in Oakland sought for the first time to 

gather large-scale epidemiological data describing oxidative damage that occurs 

in normal, healthy populations and the demographic, physical, and nutritional 

factors associated with it. 9 More than 300 volunteers (55% women, 45% men) 

between the ages of 19 and 80 were recruited, and their complete dietary information 

and medical histories were known. Plasma F 2-IsoPs were measured in all 

volunteers and statistical analyses were performed to determine whether levels 

correlated with race, sex, body mass index (BMI), vitamin intake and levels, 

and/or lipid levels. Interestingly, the strongest correlate with increasing levels of 

F 2-IsoPs was increasing BMI (Figure 3.2). 

In a second study reported in that same year, Davi and colleagues at the 

University of Rome reported a smaller study (93 volunteers) in which they 

specifically investigated lipid peroxidation in obese women by measuring levels 

of urinary F 2-IsoPs. 10 As in the first study, F 2-IsoP levels increased significantly 

with increasing BMI. Furthermore, these investigators went on to characterize 

the cause-and-effect relationship of this association by examining the effect of 

short-term, diet-induced weight loss on urinary F 2-IsoPs. Interestingly, with 

weight loss occurred a parallel decrease in BMI and a significant reduction in 

F 2-IsoP levels (Figure 3.3). Despite the small size of this weight loss study (12 

volunteers), these findings have interesting clinical implications in the possible 

role of antioxidants in the treatment of overweight and obese individuals. 

In the third and largest study by Keaney et al., urinary F 2-IsoPs were quantified 

in nearly 3000 participants in the Framingham Heart Study, and the authors again


IsoP (pg/ml) 

60 

50 

40 

30 


is up-regulated in obesity. 39 Angiotensin II has been shown to induce NADPH 

oxidase in various tissues with a resulting increase in superoxide production. 40 

Angiotensin II has also been shown to increase LDL uptake by macrophages, 

resulting in enhanced lipoprotein oxidation. 40 Further, obesity has been associated 

with reduced antioxidant defense mechanisms, including decreased erythrocyte 

glutathione and glutathione peroxidase. 41 The extent to which these and other 

mechanisms contribute to obesity-associated oxidant stress needs to be explored 

and will likely provide key information about the importance of obesity in the 

development and progression of disease. 

THERAPEUTIC TARGETS FOR REDUCING OXIDANT STRESS IN 

OVERWEIGHT AND OBESE PATIENTS 

The findings of Block, 9 Davi, 10 and Keaney 11 are not only important with respect 

to the study of basic mechanisms underlying oxidant stress associated with 

obesity, but they also have important public health implications in regard to the 

treatment of obesity-associated disease. The incidence of overweight and obesity 

is becoming more prevalent in the United States and weight loss programs are 

often ineffective. 2 Thus, the number of persons with diseases associated with 

obesity is going to be a continuing burden to the medical community 42 and novel 

strategies to prevent and treat these disorders based on the physiological perturbations 

associated with obesity need to be developed and tested. The findings of 

the studies discussed herein implicate decreasing in vivo levels of oxidant stress 

as one potential therapeutic target for obesity-associated disesase. 

ANTIOXIDANTS 

One potential therapy to reduce oxidant stress in vivo is antioxidant supplementation. 

Animal and human epidemiologic studies carried out in the 1980s and 

1990s suggested that antioxidants decreased atherosclerosis, presumably by 

reducing oxidant stress. However, prospective clinical trials of antioxidant supplementation 

using vitamin E and other agents have been disappointing and 

yielded conflicting results. 43–47 Three large trials, ATBC, GISSI, and HOPE, 

involving tens of thousands of subjects, failed to show reductions of cardiovascular 

events when vitamin E was used at doses ranging from 50 to 400 IU/day. 47–50 

On the other hand, two trials, CHAOS and SPACE, involving fewer subjects, 

reported near 50% reductions in the incidence of cardiovascular events with 

supplementation with 800 IU/day. 51,52 

All these studies suffer from the limitation of using only cardiovascular events 

as trial endpoints and not assessing oxidant stress in study participants. Thus, it 

is impossible to determine whether vitamin E or other antioxidants inhibited 

oxidant injuries in the populations studied. Small studies performed by our 

laboratory, however, have shown that in order to reduce levels of F 2-IsoPs in vivo, 

an individual must take at least 800 IU/day of vitamin E for 16 weeks and probably 

more, implying that vitamin E is a very low potency antioxidant. 53 Further,


supplementation with that very large amount of vitamin E is not practical due to 

safety issues related to consuming that amount of vitamin E. These data coupled 

with the clinical trial data suggest that supplementation with vitamin E is unlikely 

to prevent atherosclerotic events in humans. However, it is not possible to conclude 

from these studies that oxidant stress is not involved in the development 

and/or progression of atherosclerosis. Further study is needed to determine 

whether decreasing oxidative stress in vivo through antioxidant supplementation 

can prevent associated diseases. 

ω-3 POLYUNSATURATED FATTY ACIDS 

Emerging evidence has implicated increased dietary intake of fish oil containing 

large amounts of polyunsaturated fatty eicosapentaenoic acid (20:5, ω-3, EPA) 

and docosahexaenoic acid (22:6, ω-3, DHA), as beneficial in the prevention and 

treatment of a number of diseases in which environmental and lifestyle factors 

play roles. These include atherosclerotic cardiovascular disease and sudden death, 

metabolic syndrome, neurodegeneration, and various inflammatory disorders 

among others, but the mechanisms by which fish oil is protective are unknown. 54–57 

Recent data, however, suggest that the anti-inflammatory effects and other biologically 

relevant properties of ω-3 fatty acids are due, in part, to the generation 

of various bioactive oxidation products. 58–63 

One potentially important anti-atherogenic and anti-inflammatory mechanism 

of ω-3 polyunsaturated fatty acids is their interference with the arachidonic acid 

cascade that generates pro-inflammatory eicosanoids. 54,64,65 EPA can replace 

arachidonic acid in phospholipid bilayers and is also a competitive inhibitor of 

COX, reducing the production of 2-series PGs and thromboxane in addition to 

4-series leukotrienes. The 3-series-PGs and the 5-series leukotrienes derived from 

EPA are either less biologically active or inactive compared to the former products 

and are thus considered to exert effects that are less inflammatory. 66,67 Serhan and 

colleagues described a group of polyoxygenated DHA and EPA derivatives 

termed resolvins that are produced in various tissues. These compounds inhibit 

cytokine expression and other inflammatory responses in microglia, skin cells, 

and other cell types. 58–61 

In addition to studying the enzymatic oxidation of EPA, significant interest 

has focused on the biological activities of non-enzymatic free radical-initiated 

peroxidation products. Sethi and colleagues reported that EPA oxidized in the 

presence of Cu ++ , but not native EPA, significantly inhibits human neutrophil and 

monocyte adhesion to endothelial cells — a process linked to the development 

of atherosclerosis and other inflammatory disorders. 62,63 This effect was induced 

via inhibition of endothelial adhesion receptor expression and was modulated by 

the activation of the peroxisome proliferator-activated receptor-α (PPAR-α) by 

EPA oxidation products. 

Oxidized EPA markedly reduced leukocyte rolling and adhesion to venular 

endothelium of lipopolysaccharide-treated mice in vivo and the effect was not 

observed in PPAR-α-deficient mice. These studies suggest that the beneficial


effects of fish oil may be mediated, in part, by the anti-inflammatory effects of 

oxidized EPA. Similarly, Vallve and colleagues have shown that various nonenzymatically 

generated aldehyde oxidation products of EPA (and DHA) 

decreased the expression of the CD36 receptor in human macrophages. 68 Upregulation 

of this receptor has been linked to atherosclerosis. 

Additional recent reports have suggested that other related biological effects 

of fish oil, such as modulation of endothelial inflammatory molecules, are related 

to the peroxidation products of EPA and DHA. 62,69 Arita and colleagues have also 

shown that non-enzymatically oxidized EPA enhances apoptosis in HL-60 leukemia 

cells, supporting the contention that oxidized ω-3 polyunsaturated fatty 

acids are both anti-proliferative and anti-inflammatory. 70 Similar findings have 

been reported in HepG2 (human hepatoma) cells and AH109A (rat liver cancer) 

cells. 71,72 Virtually none of these reports, however, have structurally identified the 

specific peroxidation products responsible for these effects. 

The studies above that attributed some of the beneficial biological activities 

of fish oil to the oxidation of its ω-3 fatty acids provided our laboratory with a 

rationale to begin studies to systematically define the oxidation of EPA. Specifically, 

we were interested in the generation of F-ring IsoP-like compounds (F 3- 

IsoPs), structural analogs of the F 2-IsoPs containing an additional double bond 

between carbons 17 and 18, based on the hypothesis that these compounds may 

contribute to the beneficial biological effects of EPA and fish oil supplementation 

in that they may exert anti-inflammatory biological activities compared to F 2- 

IsoPs. 

One report states that the EPA-derived IsoP, 15-F 3t-IsoP, possesses activity 

that is different from 15-F 2t-IsoP in that it does not affect human platelet shape 

change or aggregation. 73 Note that 15-F 2t-IsoP is a ligand for the Tx receptor and 

induces platelet shape change and also causes vasoconstriction. 15,74 The lack of 

activity of 15-F 3t-IsoP is consistent with observations regarding EPA-derived PGs 

in that these latter compounds exert either weaker agonist or no effects in comparison 

to arachidonate-derived PGs. 55,75.76 

Anggard and colleagues provided limited evidence that F 3-IsoPs could be 

formed from the oxidation of EPA in vitro. 77 We therefore considered the possibility 

that IsoP-like compounds could be formed by the free radical-induced 

peroxidation of EPA in vivo. Our studies showed that F 3-IsoPs were generated 

from the oxidation of EPA both in vitro and in vivo. 78 Unlike results from the 

previous studies by Anggard, the structural characteristics of these compounds 

were confirmed by a number of chemical derivatization and mass spectrometric 

techniques including LC/ESI/MS/MS. 

As expected, six series of F 3-IsoP regioisomers were identified from both in 

vitro and in vivo sources. The mass spectrometric fragmentation patterns of these 

regioisomers are similar to F 2-IsoP regioisomers and, indeed, information that 

we previously acquired with F 2-IsoPs was extremely useful in the characterization 

of these molecules. 79 Of note, the relative abundance of 5- and 18-series F 3-IsoPs 

predominated over the other series. Such regioisomeric predominance was also 

reported for F 2-IsoP regioisomers in which 5- and 15-series compounds were


F 2 -IsoPs (ng/g tissue) 

75 

50 

25 

0 

− EPA + EPA 

FIGURE 3.4 Supplementation of mice with eicosapentaenoic acid (EPA) in the diet 

reduces endogenous levels of F 2-isoprostanes in the heart. 

formed in greater abundance than 8- and 12-series molecules. 79 At least part of 

the reason is likely due to the ability of precursors of 8- and 12-series F 2-IsoPs 

to undergo further oxidation and cyclization to yield a novel class of compounds 

termed dioxolane-endoperoxides. 80 Although undetermined at present, it is likely 

that a similar mechanism may account for the predominance of 5- and 18-series 

F 3-IsoPs. 

Interestingly, the levels of these compounds generated from the oxidation of 

EPA significantly exceeded those of F 2-IsoPs generated from arachidonic acid, 

perhaps because EPA contains more double bonds and is therefore more easily 

oxidizable. Furthermore, in vivo in mice, levels of F 3-IsoPs in tissues such as 

heart were virtually undetectable at baseline but supplementation of animals with 

EPA markedly increased quantities up to 27.4 ± 5.6 ng/g heart. 

Of particular note, we found that EPA supplementation markedly reduced 

levels of arachidonate-derived F 2-IsoPs by up to 64% (p


overweight and obesity contribute to disease progression. At the same time, novel 

strategies to prevent and treat these disorders based upon our understanding of 

the physiological perturbations associated with obesity need to be developed. 

The clinical studies reviewed herein by Block, 9 Davi, 10 and Keaney 11 provide 

insights into one mechanism, increased oxidant stress, that likely contributes to 

the pathophysiology of obesity-associated disease progression. Decreasing 

endogenous oxidant stress may therefore be a target for interventions for these 

diseases. Interestingly, preliminary animal studies have shown that supplementation 

with EPA, one of the major polyunsaturated fatty acids in fish oil, reduces 

in vivo levels of F 2-IsoPs, potent mediators of and important biomarkers for 

endogenous oxidant stress. Based upon these data and well-known findings that 

consumption of fish and fish oil supplementation reduce the incidence of atherosclerosis 

and other diseases in humans, fish oil consumption represents a novel 

potential treatment to decrease obesity-associated disease. 

ACKNOWLEDGMENTS 

The work reported herein was supported by National Institutes of Health grants 

GM15431, CA77839, DK48831, and ES13125. 

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n-3 and n-6 polyunsaturated fatty acids, Free Radic Biol Med 35, 189, 2003.


71. Kokura, S. et al., Enhancement of lipid peroxidation and of the antitumor effect 

of hyperthermia upon combination with oral eicosapentaenoic acid, Cancer Lett 

185, 139, 2002. 

72. Kiserud, C.E., Kierulf, P., and Hostmark, A.T., Effects of various fatty acids alone 

or combined with vitamin E on cell growth and fibrinogen concentration in the 

medium of HepG2 cells, Thromb Res 80, 75, 1995. 

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F2-alpha is not mediated by thromboxane receptor isoforms, J Biol Chem 271, 

14916, 1996. 

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F2 alpha, a potent agonist of the vascular thromboxane/endoperoxide 

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with the ovine corpus luteum prostaglandin F2 alpha receptor: implications for 

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of F3-isoprostanes during peroxidation of eicosapentaenoic acid, Biochem Biophys 

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4 

CONTENTS 


Due to Early Life 

Nutritional 

Modifications 

Malathi Srinivasan, Paul Mitrani, and 

Mulchand S. Patel 

Introduction .........................................................................................................47 

Role of Metabolic Programming in Etiology of Obesity Epidemic..................49 

Pre-Natal Metabolic Programming ...........................................................50 

Metabolic Programming Due to Altered Dietary Experience in 

Immediate Post-Natal Period ........................................................53 

High Carbohydrate (HC) Rat Model ........................................................54 

Metabolic Programming in HC Rats: Immediate Effects ........................56 

Persistent Effects in Adulthood.................................................................58 

Generational Effects ..................................................................................59 

Correlation of Altered Nutritional Experience in Early Life to 

Subsequent High Incidence of Obesity and Metabolic Syndrome ..........62 

Acknowledgment.................................................................................................63 

References ...........................................................................................................63 

INTRODUCTION 

Obesity is a rapidly growing worldwide epidemic that is especially prevalent in 

the United States. Recent data estimate that 66% of American adults are overweight, 

with about 32% being obese. 1 Interestingly, dramatic increases in obesity 

rates are relatively recent phenomena, with a rapid rise beginning in the 1980s. 2 

Between 1960 and 1980, the prevalence of obesity in the United States increased 

less than 2% (from 13 to 15%) while obesity rates in the last 25 years have more 

than doubled. 3,4 Similar trends are seen internationally, indicating that the obesity 

epidemic is a global health problem. 5 

47


Because obesity is associated with increased risks for the development of 

diabetes, cardiovascular disease, dyslipidemia, and hypertension, the epidemic of 

obesity will be followed by an epidemic of these diseases. 5,6 Therefore, it is no 

surprise that in parallel with the obesity epidemic an explosion in the incidence 

of type 2 diabetes mellitus (T2DM) has ensued. T2DM is one of the biggest 

health concerns related to obesity, affecting more than 18 million people in the 

United States and ranks as the sixth leading cause of death. 7 Between 1990 and 

1998, the prevalence of diabetes increased by 33% in the United States. 8 

Metabolic syndrome, previously referred to as syndrome X, 9 is defined by 

the clustering of cardiovascular risk factors including obesity, diabetes, hypertension, 

and dyslipidemia, 5,9 and is associated with insulin resistance. 5 Metabolic 

syndrome is now estimated to affect ~34% of American adults and up to 

36% of adult Europeans. 10 The American Medical Association estimates that 

the medical costs associated with the treatment of obesity-associated diseases 

such as T2DM and cardiovascular disorders now exceed the costs of smokingrelated 

diseases. 11 

The adult obesity epidemic has been accompanied by an analogous epidemic 

of childhood obesity, 12 especially in the United States. 13 While the prevalence of 

obesity in children has been increasing since the 1960s, the last 25 years have 

shown a rapid acceleration in childhood obesity rates. 13 During this period, the 

proportion of overweight children has almost doubled, while the proportion of 

overweight adolescents has nearly tripled, 7 leading to the increased incidence of 

T2DM in this sector of the population in the United States. 14 Additionally, the 

increase in the incidence of overweight children places them at a greater risk of 

developing obesity and T2DM as adults. 15 If the epidemics of obesity, diabetes, 

and metabolic syndrome continue to rise, the prognosis of worldwide health will 

continue to decline. Hence there is a sense of urgency in the need to further our 

knowledge about the etiology of obesity, specifically related to the steep increase 

in its incidence within the past 25 years, in an effort to curb the surge in the 

occurrence of obesity-enhanced diseases. 

The rapid rise in obesity rates since the 1980s suggests that the cause is a 

combination of various behavioral and environmental influences rather than 

specific genetic and biological changes. 1 It is commonly believed that the recent 

trends in obesity are the results of increased consumption of calorie-dense, high 

fat, and high carbohydrate foods along with decreased physical activity. 7 

Although these factors do contribute to the onset of obesity, it is now recognized 

that obesity also results from programmed impairments of energy balance that 

increase vulnerability to negative environments that include poor diet and 

limited exercise. 1 

Recent evidence suggests that both increases in food consumption and 

changes in the quality of nutrition may play decisive roles. For most of the 20th 

century, caloric consumption remained virtually constant, but then began to rise 

in the 1980s due to increased consumption of fats and sugars. 2,16 Of particular 

interest is the finding that the consumption of carbohydrates as a percentage of 

total calorie intake (due to increases in the consumption of soft drinks and fruit

Metabolic Syndrome Due to Early Life Nutritional Modifications 49 

juices) increased by approximately 20% over the past few decades. 2 In addition, 

evidence indicates that the quality of consumed carbohydrates is changing as 

high glycemic simple sugars are replacing low glycemic complex carbohydrates. 13 

Compared to high glycemic diets, low glycemic diets have been found to increase 

satiety and delay return of hunger, lower post-prandial insulin levels, and increase 

insulin sensitivity. 13 

These studies indicate that changes in the quantity and quality of dietary 

carbohydrate are important components in the pathogenesis of obesity and metabolic 

syndrome. However, in addition to changing dietary habits in adulthood, 

recent evidence indicates that exposure to altered nutrition during very early 

phases of life (pre-natal and immediate post-natal) can result in metabolic programming 

that predisposes individuals to the development of metabolic syndrome 

(Figure 4.1). 

ROLE OF METABOLIC PROGRAMMING IN ETIOLOGY OF 

OBESITY EPIDEMIC 

Population-based evidence and studies of early nutritional experiences in animals 

suggest that different nutritional insults during fetal or neonatal life may result 

in increased risks of developing metabolic diseases such as obesity and metabolic 

syndrome later in life. 10 Metabolic programming is a phenomenon in which a 

stimulus or insult that occurs during a critical period of organogenesis in early 

life results in permanent alterations in the structures and functions of affected 

organs and increased susceptibility to adult disease (Figure 4.1). 17,18 

The fetal origins hypothesis originally proposed by Barker posits that metabolic 

programming occurs in any situation in which a stimulus or insult during 

development establishes a permanent physiological response. 19 A related theory 

called the thrifty phenotype hypothesis proposes that a poor nutritional environment 

in utero results in fetal metabolic programming that maximizes uptake and 

conservation of available nutrients. 20,21 

It is well established that critical periods of development that coincide with 

periods of rapid cell division are important for the abilities of specific tissues and 

organs to differentiate and mature in preparation for survival after birth. 22,23 

Therefore, poor fetal nutrition results in selective protection of the brain at the 

expense of other organs such as the pancreas and liver, and results in altered 

growth and permanent changes in organ structure and function (Figure 4.1). 22,24 

Metabolic programming is meant to confer an adaptive advantage to an infant 

exposed to a post-natal deficiency similar to the one encountered in utero. This 

is also related to the idea of thrifty genes that are involved in the adaptive response 

to environments with scarce availability of food and function to increase survival 

in such environments. However, when these genes are introduced to situations 

where resources are abundant, they begin to lose their adaptive advantages and 

become detrimental due to increased sensitivity to and utilization of those 

resources. 20,25


FIGURE 4.1 Metabolic programming due to early life nutritional challenges. Pre-natal 

programming can be caused by maternal malnutrition, gestational diabetes, or by an 

overweight/insulin resistant pregnancy. Post-natal programming can occur due to altered 

neonatal nutrition (under- or over-nutrition, caloric redistribution). The maladaptations in 

target organs during the period of the nutritional modification persist in later life, resulting 

in increased risk for metabolic diseases in adulthood. 

PRE-NATAL METABOLIC PROGRAMMING 

Prenatal 

Programming 

Poor fetal nutrition 

- maternal malnutrition, protein deficiency, high-fat 

Gestational Diabetes 

Impairments of fetal growth, 

pancreatic development, 

and hypothalamic development 

Hormonal/metabolic abnormalities 

Metabolic Diseases in Adulthood 

(obesity, T2DM, metabolic syndrome) 

Impairments of neonatal growth, 

pancreatic development, 

and hypothalamic development 

Hormonal/metabolic abnormalities 

Inadequate neonatal nutrition 

- maternal malnutrition during lactation 

- HC dietary intervention = altered calorie sources 

Excessive neonatal nutrition 

- overfeeding (due to decreased litter sizes) 

Postnatal 

Programming 

Original studies by Barker et al. showed that adverse intrauterine conditions such 

as under-nutrition during pregnancy caused disproportionate fetal growth, resulting 

in low birth weight and adult T2DM 18,26 as well as hypertension and dyslipidemia. 

26,28 Data from several other epidemiological studies carried out in different


parts of the world support this association between fetal development in an 

undernourished maternal environment and the later onset of metabolic diseases. 29 

Although data from human epidemiological studies have indicated the connection 

between impaired fetal development and adult onset diseases such as 

T2DM, hypertension, cardiovascular problems, and other conditions, intrinsic 

difficulties surround the conduct of long-term studies in human populations. 

Therefore, several animal models have been developed to mimic the human 

situation to further our knowledge on the role of maternal nutritional status during 

pregnancy and the long-term outcome for progeny. Significant among these 

models are maternal protein or total caloric restriction and gestational diabetes 

(Figure 4.1). Several elegant reviews summarize these models in detail. 30–33 

TABLE 4.1 

Summary of Immediate and Long-Term Consequences for Progeny Due 

to Altered Maternal (Pregnancy and Lactation) Nutritional Experience 

I. Dietary protein restriction during gestation and lactation 

Immediate effects 

Fetal growth retardation 

Impairment in pancreatic islet structure and function 

Malformation of hypothalamic nuclei 

Persistent effects 

Increased insulin sensitivity in young adults 

Altered insulin signaling in peripheral tissues 

Renal defects and hypertension 

Development of glucose intolerance with advancing age 

II. Total caloric restriction during gestation and lactation 

30% caloric restriction 

Increased fasting insulin, hyperphagia, adult onset obesity and hypertension 

50% caloric restriction 

Impaired islet development, glucose intolerance 

III. Diabetes during pregnancy 

Mild diabetes 

Impaired development of fetal islets 

Impaired glucose tolerance and reduced insulin secretory capacity in adulthood 

Severe diabetes 

Fetal growth retardation resulting in smaller-sized adults 

Over-stimulation of fetal β cells due to fetal hyperglycemia 

Onset of insulin resistance in liver, muscle, and adipose tissue in adulthood 

IV. Altered intrauterine environment (maternal hyperinsulinemia/obesity) due to high 

fat diet consumption 

Fetal hyperinsulinemia and altered insulin secretory capacity 

Alterations in β and α cell volume and number on post-natal day 1 

Abnormal glucose homeostasis, impaired insulin secretion, increased body adiposity, 

hyperlipidemia, altered endothelial function and pro-atherogenic lesions in adulthood


Maternal protein malnutrition due to a low protein diet results in low protein 

(LP) progeny that are underweight, hypophagic, hypoglycemic, and hypoinsulinemic 

throughout life (Table 4.1) 34–36 and show significant alterations in pancreatic islets, 

hypothalamus, liver, and muscle (Table 4.1). 37–40 Interestingly, offspring of proteinmalnourished 

rats showed reductions in the relative weights of the pancreas, muscle, 

and liver, while the weights of the brain and lungs were protected 22,41 — consistent 

with the idea that nutrients are diverted to essential organs when scarce. 

While hypoinsulinemia in LP progeny is attributable to lower blood glucose 

levels, evidence indicates that insufficient β cell stimulation by amino acids during 

fetal development impairs insulin secretory capacity after birth. 42 Protein malnutrition 

during pregnancy reduces maternal plasma amino acid concentrations 43 

and thus placental transfer of amino acids to the fetus. 42 Reduced fetal levels of 

taurine have been associated with impaired development of normal fetal insulin 

secretion, 22,40,44,45 while supplemental taurine given to mothers on LP diets was 

able to normalize insulin secretion in fetal islets. 22,46 

Reduced insulin secretion by LP progeny is associated with reduced islet 

proliferation, size, insulin content, and vascularization, 22,34,35,44 as well as a permanent 

reduction in the number of pancreatic β cells. 35 Neonatal rats showed 

similar defects in pancreatic development when mothers were kept on low protein 

diets during lactation. 22,34 While reduced insulin secretion may confer an adaptive 

response for survival in a protein-restricted environment, the resulting impairment 

of insulin secretory capacity increases adulthood risk of glucose intolerance and 

diabetes. 22 Therefore, abnormal stimulation of β cells in early life causes malprogramming 

of β cell function that results in impaired insulin secretion throughout 

life and may increase the risk of T2DM. 

Maternal protein restriction during gestation and lactation results in offspring 

(LP) with altered programming not only of pancreatic islets but also of brain 

areas involved in the regulation of energy homeostasis and increases the risk of 

adult metabolic diseases, including decreased insulin secretion and increased 

sympathetic nervous system activity in association with hypertension. 36,47 

Among the affected brain regions are the ventromedial nucleus of the hypothalamus 

and paraventricular nucleus, which show greater relative volumes, along 

with increases in neuronal density in LP rats compared to control animals. 36 This 

increase in neuronal density is associated with increases in the activities of the 

ventromedial nucleus and paraventricular nucleus, which is consistent with data 

showing that ventromedial nucleus activity inhibits insulin secretion and stimulates 

sympathetic nervous system activity, 48 while activity of the paraventricular 

nucleus stimulates increases in blood pressure. 36,49 In addition, LP rats showed 

reduced numbers of arcuate neuropeptide Y neurons that may contribute to alterations 

in neuropeptide Y-mediated regulation of energy homeostasis including 

sympathetic nervous system activity. 36 

In the case of total caloric restriction, 50% reduction of food availability 

during the second and third weeks of pregnancy in rats resulted in impaired 

pancreatic development and glucose intolerance in the progeny at 1 year of age 

(Table 4.1). 50 Severe caloric restriction (access to only 30% of the food consumed


by controls) resulted in increased fasting plasma insulin and leptin, hyperphagia, 

hypertension, and obesity in the adult progeny. 51 

Diabetic pregnancies are associated with significant structural and functional 

changes in the fetal endocrine pancreas. For example, a mild diabetic pregnancy in 

rats resulted in hypertrophy and hyperplasia of fetal islets, 52 as well as enhanced 

proliferative capacity (Table 4.1). 53,54 In addition, fetal islets have increased insulin 

content 55 and increased responsiveness to glucose-stimulated insulin secretion. 55,56 A 

severely diabetic pregnancy, on the other hand, resulted in fetal islets that had lower 

insulin content 54 and reduced glucose-stimulated insulin secretion (Table 4.1). 56 Further, 

offspring of rat mothers with gestational diabetes showed significant increases 

in the density of arcuate neuropeptide Y neurons associated with decreased sympathetic 

nervous system activity and increased weight gain. 36,57 

Although several studies have focused on the consequences of a malnourished 

pregnancy that were important during periods of famine and war, the current dietary 

habits of people (consumption of high energy foods) especially in Westernized 

societies require investigations into the consequences for the progeny arising from 

such dietary practices in women. It has been suggested that the consumption of 

energy-dense foods is a contributing factor in the etiology of the current obesity 

epidemic and may well be responsible for a significant and increasing proportion of 

women being overweight during pregnancy. 58 Chronic feeding of a high fat diet to 

female rats bears a close resemblance to the current dietary habits of humans in 

Western societies and hence has physiological relevance to the human situation. 

Long-term consumption of a high fat diet resulted in hyperinsulinemia and increased 

insulin secretory responses to various secretogogues in term fetuses (Table 4.1). 59 

Additionally, the adult progeny of such mothers were obese, glucose-intolerant, 

hyperinsulinemic, and exhibited abnormal insulin secretory responses to glucose. 59 

Cerf et al. 60 demonstrated that feeding a high fat diet to female rats throughout 

gestation alone resulted in significant decreases in β cell volume and number and 

converse changes in α cells, resulting in hyperglycemia in 1-day-old newborn rat 

pups without changes in serum insulin concentrations. Several reports indicate 

the long-term consequences of a high fat diet during gestation only or during 

both gestation and lactation. These include abnormal glucose homeostasis, 

reduced whole body insulin sensitivity, impaired β cell insulin secretion, and 

changes in the structure of the pancreas, 61,62 defective mesenteric artery endothelial 

function, 63 hypertension, 64 alterations in conduit artery and renal functions, 65 

increased body adiposity, 61,63 deranged blood lipid profile, 61,64 hyperleptinemia, 63 

and pro-artherogenic lesions 66 in adult progeny (Table 4.1). Kozak et al. 67 demonstrated 

that a high fat diet during gestation and lactation affected body weight 

regulation in adult progeny via alterations in the functioning of neuropeptide Y. 

METABOLIC PROGRAMMING DUE TO ALTERED DIETARY EXPERIENCE IN 

IMMEDIATE POST-NATAL PERIOD 

Critical windows of development of organs and metabolic processes also extend 

into the immediate post-natal period, suggesting vulnerability of this period to


metabolic programming effects via altered nutritional experiences. In the case of 

mammals, natural rearing by mothers via breast feeding is the nature-made 

program for rearing of newborns. In humans it is well established that breast 

feeding is the optimal form of infant nutrition because it confers immunologic, 

psychological, and developmental benefits to the infant. 68,69 Several studies have 

indicated that breast feeding is protective against the development of obesity, 

diabetes, cardiovascular disease, and metabolic syndrome, 70,71 and this protection 

directly correlated with the duration of breast feeding. 72 

The association of breast feeding with decreased prevalence of childhood 

obesity 73 is thought to involve protection against metabolic disease through careful 

regulation of an infant’s calorie intake, 74 insulin secretion, 75 and adiposity. 72 

It also may help program the complex circuitry involved in the regulation of 

energy homeostasis that persists throughout life. 72 

McCance was the first researcher to demonstrate from studies in rats that 

changing the availability of milk during the suckling period had permanent effects 

on growth trajectories. 76 These studies provided the first evidence indicating a 

connection between nutritional experiences during the suckling period and longterm 

metabolic effects. Further studies stemming from adjustment of litter size 

in rats indicated that rats raised in small litters demonstrated increased body 

weight gain during the suckling period that persisted into the post-weaning period 

accompanied by hyperphagia, hyperleptinemia, hyperinsulinemia, and permanent 

alterations in gene expression and insulin secretory capacity in islets. 57,77–79 

Adult onset obesity in rats raised in small litters is supported by altered 

programming of the energy circuitry in the hypothalamus (Table 4.2). 80 Post-natal 

over-nutrition has been shown to alter the functions of the arcuate nucleus and 

ventromedial hypothalamus. For example, overfed rats showed increases in the 

density of arcuate neuropeptide Y neurons in association with increases in body 

weight and food intake. 81,82 In addition, the direction of responses to corticotropin 

releasing factor is apparently reversed in overfed rats. 83 Also in overfed rats (small 

litter size), increased inhibition of the ventromedial hypothalamic neurons by 

agouti-related peptide has been reported. 84 Rats raised in large litters during the 

suckling period maintained diminished growth patterns throughout life and 

showed reduced plasma insulin levels compared to rats from small litters of three 

or four pups per dam (Table 4.2). 76,85 

HIGH CARBOHYDRATE (HC) RAT MODEL 

Most studies on metabolic programming using animal models are involved with 

an altered intrauterine environment induced by alterations in the nutritional status 

of the mother during pregnancy. Due to difficulties in rearing newborn pups away 

from their dams, very few studies have examined the effects of nutritional modifications 

during the suckling period. Adjustment of litter size is an approach in 

this direction but results only in a decrease or increase in the availability of total 

calories during the suckling period and does not permit investigation of alterations 

in the quality of nutrition without affecting total caloric intake. In the rat, the


TABLE 4.2 

Summary of Immediate and Long-Term Consequences of Altered 

Nutritional Experience during Suckling Period 

I. Under- or over-nourishment due to adjustment of litter size 

Large-sized litters 

Reduced growth, alterations in islet functions 

Small-sized litters 

Increased growth, hyperleptinemia, and hyperinsulinemia from the start and persisting 

into adulthood 

Molecular and functional alterations in adult islets 

Alterations in hypothalamic neuropeptide expression and function 

II. Caloric redistribution (HC dietary intervention) in suckling period 

Immediate effects 

Immediate onset of hyperinsulinemia with euglycemia 

Alterations in islet insulin secretory capacity such as increased response to lower glucose 

concentrations, modified responses to incretins and neuroendocrine effectors, and ability 

to secrete moderate amounts of insulin in absence of glucose and calcium 

Molecular and structural adaptations in islets such as increased mRNA of preproinsulin 

gene, reduction in islet size and increase in numbers, increase in islet cell proliferation 

Effects observed in post-weaning period 

Persistence of hyperinsulinemia, abnormal glucose tolerance test (GTT), leftward shift 

in the insulin secretory response to a glucose stimulus, increased gene expression of 

preproinsulin and related factors, hyperphagia, significant increases in body weight gain 

and adult-onset obesity 

Generational effects 

Female rats that underwent HC dietary modification spontaneously transmitted their 

phenotypes to their progeny 

Programming effects observed in HC progeny included: 

Fetal hyperinsulinemia 

Increased response by fetal islets to glucose and amino acids 

Increased preproinsulin gene expression in fetal islets 

Absence of hyperinsulinemia during suckling period and onset of hyperinsulinemia 

immediately after weaning 

Increased response to lower glucose concentrations by islets from 28-day-old progeny 

Increases in body weight gain in post-weaning period and full-blown obesity by postnatal 

day 100 

suckling period is an important phase of continued organogenesis, including 

pancreatic and brain development. 86 

The adaptation of the artificial rearing technique originally described by Hall 87 

has enabled our laboratory to investigate the immediate and long-term consequences 

of a high carbohydrate (HC) dietary modification during the suckling 

period in rats. 88 Newborn rat pups were raised away from their dams and given 

a high carbohydrate (HC) milk formula instead of the high fat rat milk received 

by naturally reared (mother-fed; MF) rat pups. 89,90 In order to establish that the


artificial rearing technique per se did not cause any metabolic programming 

effects, newborn rat pups were also artificially reared on a high fat (HF) milk 

formula, the macronutrient composition of which was identical to that of rat 

milk. 90,91 Although MF, HF, and HC rat pups received a similar number of calories 

per day, the primary source of calories was switched from fats to carbohydrates 

for the HC rats. The mere change in the quality of nutrition during the suckling 

period (from fat-enriched rat milk to carbohydrate-enriched HC milk formula) 

resulted in metabolic programming due to the overlap of the HC dietary intervention 

and the critical post-natal period of pancreatic development in rats. 88,92,93 

The high carbohydrate nature of the HC formula (56% carbohydrate compared 

to 8% in rat milk and in the HF milk formula) increases insulin demand in neonatal 

rats. This altered nutritional environment induces compensatory adaptations in 

islet function to ensure that the immediate demand for insulin is met. However, 

permanent programming of these adaptations results in altered responses to nutritional 

experiences throughout life and adverse consequences in adulthood (Table 

4.2). 88,92,93 

METABOLIC PROGRAMMING IN HC RATS: IMMEDIATE EFFECTS 

Artificial rearing of rat pups on the HC milk formula resulted in the immediate 

onset (within the first 24 hours) of hyperinsulinemia when compared to MF and 

HF (experimental control) rats and persisted during the entire suckling period in 

the HC rats (Figure 4.2). 88,94 Interestingly, HC pups showed normal blood glucose 

levels during this period despite hyperinsulinemia, suggesting that the alterations 

in islet function are able to compensate for increased insulin demand to maintain 

0 22 4 

Birth 

12 


Cellular adaptations: changes in islet architecture 

Molecular adaptations: increases in preproinsulin 

gene transcription and related transcription 

factors 

Biochemical adaptations: increased response of 

isolated islets to various stimuli 

Hyperphagia and 

Significant increases 

in body weight and an 

upward trend in abnormal response to a 

body weight gain glucose load Full blown 

HC 

obesity 

Prenatal Suckling Post-weaning 

24 40 55 75 100 

Postnatal age (days) 

FIGURE 4.2 Overview of immediate and lasting consequences of HC dietary modification 

in the immediate post-natal period in first generation HC rats.


euglycemia. 94 In contrast, rats artificially reared on an HF formula (caloric distribution: 

carbohydrate 8%, protein 24%, and fat 68%) as an internal control for 

the artificial rearing technique were not hyperinsulinemic, suggesting that the 

metabolic programming of HC rats was the result of a change in the quality of 

nutrition and not a change in the mode of delivery of nutrition during the suckling 

period. 90 

In vitro studies of glucose-stimulated insulin secretion (GSIS) in isolated 

pancreatic islets showed that neonatal HC islets secreted significantly more insulin 

after both 10 and 60 min of glucose stimulation compared to MF islets at all 

glucose concentrations studied. 94 In association with this increased insulin secretory 

response to glucose, HC islets showed increases in (1) the enzyme activities 

of the low K m hexokinase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate 

dehydrogenase complex and (2) increases in levels of glucose transporter 

protein-2 (Figure 4.2). 94 Up-regulation of glucose metabolism in HC islets suggests 

that a lowered glucose threshold for insulin secretion may, in part, be 

responsible for the development of hyperinsulinemia due to programmed hypersensitivity 

of pancreatic islets to glucose. 

In vivo insulin secretion is stimulated by numerous secretogogues including 

glucose, amino acids, fatty acids, and neuroendocrine and incretin factors. 95 While 

insulin secretion is primarily induced by glucose stimulation of the K ATP channeldependent 

pathway, acetylcholine, norepinephrine, glucagon, glucagon-like peptide-1, 

and other signals act on K ATP channel-dependent and -independent pathways 

as well as Ca 2+ channel-independent pathways to augment insulin secretion. 

95 Plasma glucagon-like peptide-1, an incretin hormone that promotes insulin 

secretion via increases in cAMP levels, 96 was increased in 12-day-old HC rats as 

were glucagon-like peptide-1 receptor mRNA levels in isolated HC islets. 97 Acetylcholine 

and glucagon-like peptide-1 potentiation of glucose-stimulated insulin 

secretion was greater in HC islets compared to MF islets, indicating increased 

responsiveness to these agonists. 97 Also, the activities of protein kinase C, protein 

kinase A, and calcium calmodulin kinase II were significantly higher in HC islets 

compared to MF islets, 86 as were the mRNA levels of adenylyl cyclase type VI. 97 

Furthermore, HC islets have reduced sensitivity to norepinephrine stimulation, 

which inhibits insulin secretion through activation of α-adrenergic receptors on 

β cells 98 compared to control islets. 97 

In addition to adaptations at the level of the insulin secretory process, molecular 

adaptations were observed in islets isolated from 12-day-old HC rats. The 

adaptations included significant increases in insulin biosynthesis and in the 

mRNA levels of the preproinsulin gene and other components of the cascade 

implicated in the regulation of the transcription of the preproinsulin gene in 

neonatal HC islets (Figure 4.2). 99 Gene array analysis of the mRNAs from neonatal 

HC islets indicated that several clusters of genes involved in a wide array 

of cellular functions (e.g., cell cycle regulation, protein synthesis, ion channels, 

and metabolic pathways) were up-regulated in these islets and may contribute to 

the onset of hyperinsulinemia in these rats. 100


The overlap of the HC dietary modification with the period of post-natal 

development of the pancreas also resulted in several changes in the cellular 

architecture of neonatal HC islets (Table 4.2; Figure 4.2). These include: (1) a 

reduction in mean islet size and increase in the number of small-sized islets, (2) 

an increase in the number of islets per unit area, (3) greater apoptotic rates in 

islets compared to ductal epithelium, and (4) increases in islet cell replication 

as indicated by the presence of proliferating cell nuclear antigen in a large 

proportion of β cells in 12-day-old HC islets compared to islets from agematched 

MF rats. 101 

Metabolic programming effects were also observed in livers of neonatal HC 

rats and may be important for the establishment of the HC phenotype observed 

in adulthood. Precocious induction of the activities of glucokinase and malic 

enzyme were indicated by substantial increases in their activity levels in 10-dayold 

HC animals. 89 The lipogenic capacity and glycogen content were also 

increased in the livers of these rats. 89,102 

The hypothalamus is an important site for energy homeostasis and aberrations 

in the expression and function of a network of neuropeptides lead to either 

hyperphagia or hypophagia. 103,104 HC dietary modification resulted in significant 

increases in the protein content and gene expression of orexigenic signals and 

converse changes in the anorexigenic signals, suggesting that alterations leading 

to the eventual onset of obesity in the adulthood of these rats are evident very 

early in life (M.S. Patel, unpublished observations). 

PERSISTENT EFFECTS IN ADULTHOOD 

HC rats were hyperinsulinemic throughout the period of nutritional modification 

and continued to be hyperinsulinemic into adulthood even after weaning onto 

laboratory rodent diets on post-natal day 24 (Figure 4.2). 90,91 Studies of 100-dayold 

adult rats showed that HC animals had significantly higher plasma insulin 

levels compared with age-matched MF rats. 105 In addition to persistent hyperinsulinemia, 

adult HC rats showed impaired glucose tolerance compared with 

controls, even though they were euglycemic, suggesting a state of insulin resistance 

(Figure 4.3). 91 

As in the case of 12-day-old HF rats, adult HF rats were not hyperinsulinemic 

and did not show altered growth or food intake compared to MF rats. 90 While 

HC and MF rats showed similar body weights during the suckling period, 94 HC 

rats experienced increased growth rates after weaning in association with hyperphagia 

that resulted in full-blown obesity by day 100 (Figure 4.2). 90,106 

In addition, islets from adult HC rats showed increases in GSIS, low K m 

hexokinase activity, and Ca 2+ -independent insulin release similar to results from 

12-day-old HC islets. 105 Additionally, increases in insulin producing cell masses 

in adult HC pancreas were noted. 91 At the molecular level, increased preproinsulin 

gene transcription with concomitant increases in mRNA levels of transcription 

factors regulating its gene expression and increases in the expression of several 

clusters of genes as indicated by gene array analysis 105 were observed in


Plasma glucose (mM) 

Plasma glucose (mM) Plasma glucose (mM) 

16 

12 

8 

4 

20 

16 

12 

8 

24 

20 

16 

12 

8 

FIGURE 4.3 Plasma glucose and insulin levels in HC male rats after an oral glucose 

tolerance load (2 g/kg body weight) on post-natal days 64, 78, and 270. 91 Completely 

overlapping points for plasma glucose of 270-day old MF and HC rats are shown with 

open circles only. 

100-day-old HC islets, suggesting that the molecular effects observed in neonatal 

islets persist into adulthood. Adult onset obesity was accompanied by increases 

in the lipogenic capacities of the liver and epididymal adipose tissue, increases 

in the epididymal adipose tissue weight, and marked increases in the cell sizes 

of epididymal and omental adipose tissues of 100-day-old male HC rats. 90 

GENERATIONAL EFFECTS 

Day 64 

0 

0 20 40 60 80 100 120 0 20 40 60 80 100 120 

Day 78 

Day 270 

A notable observation from the HC model is that the progeny of HC female rats 

that underwent the HC dietary modification in their immediate post-natal life 

Plasma insulin (pM) 


800 

600 

400 

200 

1600 

1200 

MF 

HC 

0 

0 20 40 60 80 100 120 0 20 40 60 80 100 120 


800 

400 

1600 

1200 

0 20 40 60 80 100 120 0 20 40 60 80 100 120 

Time (min after glucose bolus) Time (min after glucose bolus) 

800 

400


Term fetus 

Hyperinsulinemia, normoglycemia 

Increased insulin secretory response 

by islets 

Increased expression of preproinsulin 

gene 

Maternal 

hyperinsulinemia, 

increased body 

weight gain during 

pregnancy 

Prenatal Suckling Post-weaning 

0 210 12 24 28 55 75 10 

Term fetus 


Increased sensitivity to basal glucose 

Increased expression of preproinsulin 

gene 

Increases in body 

weight gain 

Postnatal age (days) 

Full blown 

obesity 

FIGURE 4.4 Overview of generational effects in progeny (second generation HC rats) 

resulting from HC dietary modifications in female rats. 

spontaneously acquired the HC phenotype (chronic hyperinsulinemia and adult 

onset obesity) without undergoing dietary modifications (Figure 4.4). 106 The 

intrauterine environment in the HC female rat was characterized by hyperinsulinemia 

and normoglycemia. 106–108 We recently determined that the HC female rats 

consumed significantly increased amounts of food and gained significantly more 

weight during gestation compared to pregnant MF controls; they were hyperleptinemic 

on gestational day 21 (M.S Patel, unpublished observations). 

HC term fetuses (gestational day 21) were hyperinsulinemic and normoglycemic. 

Fetal hyperinsulinemia was accompanied by increased pancreatic insulin 

content, increased gene expression of preproinsulin and pancreatic duodenal 

transcription factor-1, and increased insulin secretory responses to various secretogogues 

(Figure 4.4). 108 Although no significant changes were noted in plasma 

insulin levels during the suckling period between the second generation HC (2- 

HC) and MF rats immediately after weaning, the plasma insulin levels of 2-HC 

rats were significantly increased (first observed on post-natal day 26; Figure 

4.4). 107 As observed in term fetuses, there was a leftward shift in the insulin 

secretory response to a glucose stimulus and increased preproinsulin gene transcription 

in islets from 28-day old 2-HC rats. 107 

Similar to the observations in first generation HC rats, no significant differences 

were noted in the body weights of 2-HC rats compared to age-matched 

MF rats up to post-natal day 55. After post-natal day 55, significant increases in 

body weight were observed with full blown obesity evident on approximately 

post-natal day 100. 106 This correlates with significant increases in the weight of 

the adipose tissues, increases in the size of the adipocytes, and increases in the 

activities of the lipogenic enzymes (fatty acid synthase and glucose-6-phosphate 

dehydrogenase) in both liver and adipose tissue of 100-day old 2-HC rats. 106


A state of insulin resistance was indicated by decreased content of glycogen 

in livers and muscles of 100-day-old 2-HC male rats. 109 The decrease was associated 

with a decrease in the enzyme activity of glycogen synthase and its putative 

upstream activators. 109 In contrast, the situation was reversed in the epididymal 

adipose tissues of these rats. 110 

Are the metabolic programming effects induced by early life HC dietary 

modifications in rat pups reversible phenomena? In order to address this question, 

we pair-fed HC female rats from the time of weaning to the amount of feed 

consumed by age-matched MF female rats on a daily basis. Interestingly, such a 

dietary control in HC female rats resulted in reductions of their plasma insulin 

levels and body weight gains to the levels observed in age-matched female rats. 108 

During pregnancy, the plasma insulin levels and body weights were similar in 

the pair-fed HC and age-matched MF female rats. 108 Such improvements in 

pregnancy conditions in the pair-fed HC female rats prevented the transmission 

of the HC phenotype to their progeny. 108 These results indicate that dietary 

regulation in HC rats results in the reversal of the metabolic programming effects 

with good prognosis both for rats of the same generation and the subsequent 

generation. 

The mechanisms responsible for metabolic programming due to altered nutritional 

experiences in early life are not well understood. Malorganization (functional 

and/or structural) of target organs has been suggested as a plausible mechanism. 

111 In animal models for pre-natal metabolic programming (protein 

malnourishment, gestational diabetes, and caloric restriction), such adaptations 

in target organs were reported. 30 In the HC rat model, we observed functional 

and/or structural changes in neonatal HC rats in target organs such as the islets, 

gut, hypothalamus, and liver. All of these organs are potentially important for 

maintaining glucose homeostasis. As indicated in Figure 4.5, it is possible that 

adaptations in the these organs and the resultant cross-talks among them are 

necessary for survival of these rat pups in the face of the HC dietary modification 

but since they occur during the period of immediate post-natal development, such 

adaptations become permanent and in the post-weaning period, predispose these 

animals to adult onset obesity and concomitant health issues. 

Various interactions between target organs are suggested in Figure 4.5. The 

HC milk formula may induce alterations in stomach- and gut-derived factors 

which, through specific receptor-mediated signaling, may alter the functional 

capabilities of organs such as the islets and the hypothalamus. Similarly, insulin 

via receptors present in the islets and the hypothalamus may exert altered autocrine 

and endocrine functions to support the HC phenotypes in these rats. Several 

neuropeptides enumerated in the hypothalamus have principal roles in the energy 

homeostasis of the body, insulin secretion by islets, insulin sensitivity of target 

organs, etc. Therefore, a multifactorial mechanism may be responsible for the 

onset and persistence of hyperinsulinemia in HC rats which predisposes these 

rats for insulin resistance leading to altered glucose tolerance and obesity in 

adulthood.


Brain (hypothalamus) 

Alterations in energy 

homeostasisrelated 

neuropeptides 

Diet 

(high carbohydrate) 

Pancreas 

(islets) 


(immediate response) 

Stomach and gut 

(gut-related factors) 

Metabolic adaptations in peripheral tissues 

Persistence of hyperinsulinemia 

Obesity and other complications in adulthood 

Unfavorable intrauterine environment in female rats 

Transmission of the maternal phenotype to the progeny 

(perpetuation of the metabolic programming effects) 

FIGURE 4.5 Postulated scheme leading to development of the HC metabolic phenotype 

in adulthood due to high-carbohydrate dietary modifications in neonatal rats. The direct 

effects of the dietary treatment on target organs and possible cross-talks among these 

organs leading to the adult phenotype are indicated. 

CORRELATION OF ALTERED NUTRITIONAL EXPERIENCE IN 

EARLY LIFE TO SUBSEQUENT HIGH INCIDENCE OF OBESITY 

AND METABOLIC SYNDROME 

As indicated, nutritional experiences in utero and/or in the immediate post-natal 

period (infancy) can to a significant degree influence adult metabolic phenotype 

as these are periods of rapid development of the organism. Because the main goal 

is survival, an organism adapts to the altered environment by necessary alterations 

in target tissues that help it “tide over” the situation but such adaptations lead to 

unfavorable situations later in life. Both epidemiological data and results from 

animal studies indicate the importance of adequate nutrition during pregnancy. 

Studies of the long-term consequences of an altered nutritional experience in 

early post-natal life indicate the importance of this phase of life for metabolic 

programming effects. Dietary habits for all ages have undergone tremendous 

changes over the past several decades. The present obesity epidemic, to a large 

measure, may be the result of such changes. Extrapolation of data obtained from 

HC rat models suggests that post-natal increased consumption of carbohydrates


by infants (formula feeding with early introduction of carbohydrate-rich supplements 

such as cereals, fruits juices, etc.) in Western societies may be partly 

responsible for the increase in the incidence of obesity. This effect is exacerbated 

by the mode of feeding (bottle, spoon, etc., resulting in overfeeding). Supplementation 

of milk (breast or formula) with early introduction of carbohydrateenriched 

baby foods and overfeeding may result in malprogramming effects in 

these babies, leading to adult onset obesity and attendant diseases as observed in 

the HC rat model. 

The spontaneous transfer of the HC phenotype to the progeny and results 

from our studies of maternal high-fat diet-induced effects in the next generation 

indicate that maternal obesity primes obesity in progeny. Our results suggest that 

females that are overnourished or exposed to increased carbohydrate intake in 

infancy may be obese and insulin resistant during pregnancy and, due to an 

unfavorable intrauterine environment, are at increased risk for the establishment 

of a vicious cycle of transmission of their metabolic phenotype to their progeny. 

The alarming increase in the numbers of overweight and obese individuals 

in the United States suggests that a significant number of pregnancies will not 

be under conditions of optimal health due to increased body weight, moderate 

hyperinsulinemia, and mild insulin resistance in these females. Fetal development 

under such conditions may predispose the progeny for the development of obesity 

in adulthood. This generational effect may be amplified by dietary habits in 

infancy and lifestyle later in adulthood such that the maternal intrauterine environment 

becomes more and more favorable for metabolic malprogramming of 

the progeny from one generation to the next. 

ACKNOWLEDGMENT 

This work was supported in part by National Institute of Diabetes and Digestive 

and Kidney Diseases grant DK-61518. 

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lead to chronic disease, Am. J. Clin. Nutr. 69, 179, 1999.

5 

CONTENTS 

Oxidative Stress and 

Antioxidants in the 

Perinatal Period 

Hiromichi Shoji, Yuichiro Yamashiro, and 

Berthold Koletzko 

Introduction .........................................................................................................71 

Oxidative Stress ..................................................................................................72 

Pregnancy: Oxidative Stress ...............................................................................74 

Preeclampsia..............................................................................................75 

Oxidative Stress at Childbirth.............................................................................75 

Perinatal Asphyxia.....................................................................................76 

Use of 100% Oxygen in Resuscitation.....................................................77 

Oxidative Stress in Premature Infants ................................................................78 

Bronchopulmonary Dysplasia ...................................................................79 

Neonatal Necrotizing Enterocolitis ...........................................................80 

Periventricular Leukomalacia....................................................................80 

Retinopathy of Prematurity .......................................................................81 

Human Milk and Antioxidative Protection.........................................................82 

Conclusions .........................................................................................................84 

Acknowledgments ...............................................................................................84 

References ...........................................................................................................85 

INTRODUCTION 

Oxidative stress is thought to be implicated in many pathologic processes of 

human disorders. 1 Pregnancy is a physiological state of oxidative stress accompanied 

by high metabolic demands and elevated requirements for tissue oxygen. 2 

Childbirth is also accompanied by increased oxidative stress. Numerous disorders 

in infancy have been linked with oxidative stress, while the role of oxidative 

stress in the pathogenesis and progression of these diseases is only partially 

defined. 3 In 1988, Saugstad advocated “oxygen radical disease in neonatology” 

as a unifying hypothesis for the role of oxidative stress in a wide range of neonatal 

71


morbidities, implying different manifestations of the same condition. 4 Thus it has 

become important for clinicians who treat patients in the perinatal period to 

understand oxidative stress. The purpose of this chapter is to review information 

about the role of oxidative stress in pregnancy and post-partum. 

OXIDATIVE STRESS 

Free radicals are highly reactive chemical molecules containing one or more 

unpaired electrons. They donate or take electrons from other molecules in an 

attempt to pair their elections and generate a more stable species. Reactive oxygen 

species (ROS) is a collective term that includes both the oxygen free radicals (the 

O 2· – superoxide and the OH· hydroxyl radical) and some non-radical (hydrogen 

peroxide, H 2O 2) derivatives of oxygen. ROS are normally produced in living 

organisms with the potential of reacting with almost all types of molecules in 

living cells. ROS may be generated by different mechanisms such as 

ischemia–reperfusion, neutrophil and macrophage activation, Fenton chemistry, 

endothelial cell xanthine oxidase, free fatty acid and prostaglandin metabolism, 

and hypoxia (Figure 5.1) .5,6 

Mitochondrial respiration is also one of the main physiological sources for 

the generation of ROS. Superoxide is formed when electrons leak from the 

electron transport chain. 7 ROS are capable of damaging all biologic macromolecules 

including lipids, proteins, polysaccharides, and DNA. When ROS are 

overproduced, they are important mediators of cell and tissue injury 8,9 and may 

cause cell death by apoptotic and necrotic mechanisms. 10 

• Electron transport chain 

(mitochondria) 

• NADPH oxidase 

(neutrophil activation) 

• Xanthine oxidase 

• Lipid radicals 

(fatty acid metabolism) 

O 2 

NO 

.− O2 H 2 O + O 2 

GPx, CAT 

SOD 

ONOO − 

H 2 O 2 

FIGURE 5.1 Reactive oxygen species produced in tissues. 

Fenton reaction 

Fe 2+ 

OH. 

Oxidation of DNA, 

proteins, and lipids

Oxidative Stress and Antioxidants in the Perinatal Period 73 

TABLE 5.1 

Major Antioxidants 

Enzymes Superoxide dismutase 

Catalase 

Glutathione peroxidase 

Glutathione reductase 

Non-enzymatic 

antioxidants 

Vitamins Vitamin E 

Vitamin A 

Vitamin C 

Coenzyme Q 

β-carotene 

Reducing agents Glutathione 

Cysteine 

Thioredoxin 

Binding proteins Albumin 

Ceruloplasmin 

Lactoferrin 

Transferrin 

Enzyme constituents Zinc 

Selenium 

Others Uric acid 

Bilirubin 

Erythropoietin 

A more recently appreciated group of molecules known as reactive nitrogen 

species are derived from reactions involving another free radical, nitric oxide 

(NO). NO can act either as a pro-oxidant or as an antioxidant. 11 O 2· – reacts with 

NO to form a cytotoxic oxidant peroxynitrite (ONOO – ) (Figure 5.1). 

Oxidative stress can be defined as an imbalance between the amount of ROS 

and the intracellular and extracellular antioxidant protection systems. 12 An antioxidant 

may be classified as “any substance that can delay or prevent oxidation 

of a particular substrate” (Table 5.1) 13 and it may be broadly classified as enzymatic 

(superoxide dismutases; SOD, catalase; CAT, glutathione peroxidase; GPx 

and glutathione; GSH and their precursors) or non-enzymatic. 14 The antioxidant 

defense can be divided into extracellular and intracellular defenses that protect 

against ROS-induced cell damage. Transferrin, ceruloplasmin, vitamin C, vitamin 

E, uric acid, bilirubin, sulfhydryl groups, and other unidentified antioxidants 

contribute to the total antioxidant capacities of extracellular fluids. 15 

The extent of oxidative stress has been variably determined by measurement 

of a decrease in total antioxidant capacity, through depletion of individual antioxidants 

such as vitamin E, vitamin C, or GPx. Otherwise it is defined as the 

product (marker) of oxidative stress to lipids, proteins, and DNA.


PREGNANCY: OXIDATIVE STRESS 

Pregnancy accompanied by a high metabolic demand and elevated requirements 

for tissue oxygen represents a physiological state of oxidative stress. 2 The placenta 

has been identified as an important source of lipid peroxidation because of its 

enrichment with polyunsaturated fatty acids (PUFAs). 16 Falkay et al. suggested 

that the increase in the lipid peroxidation levels is due to increased prostaglandin 

synthesis in the placenta. 17 Levels of peroxidation markers such as lipid hydroperoxide 

and malondialdehyde (MDA) are higher in pregnant than in non-pregnant 

women. 18 Lipid peroxidation is enhanced in the second trimester, tapers off 

later in gestation, and decreases after delivery. 19 

A certain amount of ROS promotes embryonic development. 20–22 ROS seem 

to play a role in signal transduction by modulating transcription factors including 

hypoxia-inducible factor (HIF-1) 23 and activated protein-1 (AP-1) 24 that control 

the expression of cell growth mediators. ROS may also affect activation and 

release of the nuclear factor NFκB — an important regulator of cytokine and 

anti-apoptotic gene expression. 23,25,26 However, over-production of ROS may also 

induce early embryonic developmental block and retardation (Figure 5.2). 20,23,27 

The placenta is also a source of antioxidative enzymes to control placental 

lipid peroxidation during uncomplicated pregnancies. All the major antioxidant 

systems including SOD, CAT, GPx, glutathione S-transferase, and GSH, and 

vitamins C and E are present in the placenta. 28–31 The activities of SOD and CAT 

increased as gestation progressed, while the activity of GPx and vitamin E 

concentration did not significantly change. 29,32,33 One study found that placental 

concentration of lipid peroxidation decreased as gestation advanced, with 

increased activities of SOD and CAT in the placenta. 34 Placental antioxidant 

defense systems may be sufficient to control the lipid peroxidation in normal 

pregnancies. 35,36 

Embryonic cell 

Transcription factor activation 

(HIF-1, AP-1, NF-κ B) 

Reactive oxygen species 

Oxidative stress 

Apoptosis/necrosis 

Promote cell development Impair cell development 

FIGURE 5.2 Role of reactive oxygen species in fetal development. (Source: Modified 

from Dennery, P.A. Antioxid. Redox Signal. 6, 147, 2004.)


Abnormal placentation (inadequate placental invasion of the maternal spiral 

arteries) leads to reduced uteroplacental blood flow and placental ischemia. 37 The 

ischemia–reperfusion to the placenta leads to abnormal generation of placental 

oxidative stress. Excessive placental oxidative stress was assumed to play a role 

in the pathogenesis of preeclampsia (PE) 16,38,39 and intrauterine fetal growth 

retardation (IUGR). 39–42 

PREECLAMPSIA 

Preeclampsia (PE) is a pregnancy-specific disorder that complicates 5% of all 

pregnancies and 11% of all first pregnancies. 42 Clinically, PE is usually diagnosed 

in late pregnancy by increased blood pressure and proteinuria, and the symptoms 

of PE typically disappear shortly after delivery of the placenta. The placenta plays 

a major role in the pathogenesis of PE, characterized by abnormal placentation 

and reduced placental perfusion. 43 Recent evidence suggests that a disturbance 

of normal endothelial cell function may be a primary cause in the pathogenesis 

of PE. 44,45 PE is associated with severe maternal and fetal mortality 46 and is a 

major risk factor for preterm delivery and IUGR. 47 

Placental oxidative stress resulting from ischemia–reperfusion injury is 

involved in the pathogenesis of PE. A significant increase in placental lipid 

peroxidation levels in the placenta of PE has been demonstrated. 48–50 Several 

reports confirm that circulating levels of lipid peroxides are significantly elevated 

in women with PE compared to normal pregnant women. 18,51–53 However, other 

studies showed no evidence of increased circulating lipid peroxidation in PE. 54–56 

Lluba et al. speculated that circulating lipid peroxides generated in preeclamptic 

women are neutralized by the increasing concentration of antioxidant 

enzymes and vitamin E. 55 On the other hand, several important antioxidants are 

significantly decreased in pregnant women with PE. The levels of vitamin C, 

vitamin E, vitamin A, β-carotene, coenzyme Q10, and GSH are all significantly 

lower than in normal pregnancy. 52,53,57,58 Although most studies report decreased 

levels of vitamin E in preeclamptic women, some do not. 54,55 

Vitamin C and vitamin E have been studied related to prevention of PE. Early 

intervention at 16 to 22 weeks of pregnancy (283 women) with vitamin C (1000 

mg/day) and E (400 IU/day) supplementation resulted in significant reductions 

of PE in the supplement group 59 but a recent report of a randomized trial (100 

women) failed to reveal the benefits of PE prevention after the same amounts of 

vitamins C and E were supplemented at 14 to 20 weeks of gestation. 60 

OXIDATIVE STRESS AT CHILDBIRTH 

Childbirth is an oxidative challenge for newborns. The transition from fetal to 

neonatal life at birth implies acute and complex physiologic changes. The fetus 

transfers from an intrauterine hypoxic environment with a pO 2 of 20 to 25 mm 

Hg to an extrauterine normoxic environment with a pO 2 of 100 mm Hg. 61 This 

four- to five-fold increase is believed to induce increased production of ROS.


Neonatal plasma has relatively poor antioxidative defenses. 62 At birth, neonatal 

plasma concentrations of vitamins A, E and β-carotene were significantly 

lower than maternal plasma levels, while neonatal levels of vitamin C were 

significantly higher. 63 Uric acid and vitamin C constitute most of the extracellular 

antioxidant capacity, totaling 75%. At 2 weeks of age, these two components 

represent only 35% of the extracellular capacity. This change is caused by the 

rapid decline of vitamin C levels during the first few days of life and the increasing 

concentration of bilirubin which acts as an antioxidant in the first 1 to 2 weeks 

post-partum. 64 

Term labor is associated with oxidative stress for the neonate. Reported MDA 

levels were higher for term infants born by cesarean section than for those born 

by spontaneous vaginal delivery. 65,66 In a case controlled study, the serum levels 

of lipid peroxidation products were significantly higher (110%) in 20 women 

during labor compared to the 20 controls (pregnant women not in labor and 

matched for maternal gestational age). 67 On the other hand, women during term 

labor showed up-regulations of red blood cell GSH in cord blood. Fetal (difference 

in GSH concentration in umbilical vein and artery) and maternal (pre- and postdelivery) 

GSH concentrations were significantly lower during labor at term than 

in elective cesarean section. 68 

PERINATAL ASPHYXIA 

Perinatal asphyxia is characterized by transient hypoxia during the ischemic phase 

followed by reperfusion. One definition of birth asphyxia is based on the finding 

of three of the following four criteria: (1) pH of umbilical arterial cord blood 


ATP 

Ischemia 

Tissue damage 

Denosine 

Inosine 

Hypoxanthine 

Reperfusion (restores O 2 ) 

− Xanthine, O , H2O 2 2 

Fe 2+ /Cu 2+ 

FIGURE 5.3 Suggested mechanism for tissue damage upon reperfusion of hypoxic and 

ischemic tissues. (Source: Modified from Halliwell, B. and Gutteridge, J.M., Free Radicals 

in Biology and Medicine, Oxford University Press, New York, 1999.) 

approached term. 40 ROS cause endothelial cell damage and abnormalities in Nmethyl-D-aspartate 

receptors, synaptosome structures, and astrocyte functions, 

thus contributing to the development of brain injury after a hypoxic–ischemic 

episode. 74–76 

USE OF 100% OXYGEN IN RESUSCITATION 

Xanthine dehydrogenase 

Xanthine oxidase 

The optimal concentration of oxygen for neonatal resuscitation is uncertain. 

Traditionally, neonatal resuscitation has been performed with 100% oxygen. 

Many textbooks and the advisory statement of the International Liaison Committee 

on Resuscitation 77 recommend that resuscitation of newborn infants should 

be performed with 100% oxygen. Resuscitation of depressed newborns with 

presumed hypoxia and/or asphyxia in the delivery room is currently the only 

remaining clinical indication for the use of unregulated 100% oxygen in infants. 

Exposure to high levels of oxygen during reoxygenation may promote the 

formation of excessive levels of ROS in tissue, causing tissue injury. Hyperoxemia 

has been associated with numerous negative side effects including delayed initiation 

of spontaneous respiration, increased oxygen consumption, and irregularities 

in cerebral circulation. 78 Therefore, several groups have investigated the safety 

and efficacy of using room air for neonatal resuscitation. 

Evidence from animal studies proves that room air is as effective as 100% 

oxygen in resuscitation. No significant differences were noted between two 

groups (hypoxemic newborn pigs resuscitated with 21% O 2 or 100% O 2 for 20 

to 25 min followed by 21% O 2) in arterial blood pressure, base deficit, plasma 

OH −


GSH/GSSG 

80 

60 

40 

20 

Nonasphyxiated neonates 

RAR 

OxR 

0 

0 3 28 

Days of postnatal life 

FIGURE 5.4 Reduced gluthatione (GSH) and oxidized gluthathione (GSSG) ratio in 

asphyxiated neonates resuscitated with 100% oxygen (OxR) or room air (RAR). ** p 


antioxidant synthesis and activities) are low in preterm infants compared to term 

infants. 88,89 Induction of antioxidative enzymes following oxidative stress was 

found in term but not in preterm infants. 90 These data strongly suggest increased 

susceptibilities of premature infants to oxidative stress. 62 Clinical conditions in 

which oxidative stress may be particularly relevant in premature infants are 

discussed below. 

BRONCHOPULMONARY DYSPLASIA 

Ventilated premature infants are at risk of developing bronchopulmonary dysplasia 

(BPD), a chronic lung disease of prematurity. When BPD was first described, 

exposure to a high oxygen level was identified as a risk factor for its 

development 91,92 and it was soon related to ROS. 93 It is widely assumed that BPD 

is due to ROS-related injury to the immature lung. 94 Two studies found a close 

relationship between high oxygen exposure and the development of BPD. 95,96 Van 

Marter et al. found that inspired oxygen concentrations were higher in infants 

who developed BPD compared with those who did not. A fractional inspired 

oxygen level of 1.0 in the first day of life almost doubled the risk of BPD relative 

to an FiO 2. 96 Oxidative stress detected by the augmentation of lipid peroxidation 

products in the very first days of life is associated with the subsequent development 

of BPD. 97,98 

Antioxidants present in the alveolar epithelial lining fluid are well positioned 

to protect against tissue damage caused by ROS generated by these mechanisms. 99 

Infants who develop BPD have decreased concentrations of glutathione in bronchoalveolar 

fluid compared to those who did not require supplemental oxygen at 

36 weeks post-conceptional age on the first day of life. 86 Robbins et al. reported 

that the administration of recombinant human SOD (rhSOD) in newborn piglets 

mitigated the lung inflammatory changes (analyzed by BAL neutrophil chemotactic 

activity and cell count), MDA concentration in lung tissue, and acute lung 

injury induced by 100 ppm of NO and 90% O 2. 100 In a multicenter controlled 

study of 26 preterm infants, significant reductions in radiological evidence of 

BPD were noted in infants treated with rhSOD compared to infants treated with 

placebo. 101 However, in a later and larger study of 302 infants, no differences in 

the development of BPD in infants treated with rhSOD and those treated with 

placebo were noted. 102 

NO is a free radical that may be oxidized or reduced, depending on its 

concentration and the presence of other oxidants such as oxygen. Hyperoxic 

exposure of rat pups up-regulated both inducible and endothelial NO synthase 

and therefore increased the concentrations of NO and subsequently peroxynitrite 

as well. 103 NO is highly toxic for preterm infants. However, a clinical trial with 

inhaled NO did not report higher occurrences of BPD in NO-treated premature 

infants compared with controls. 104 Hamon et al. recently reported that low doses 

(5 ppm) of inhaled NO administered soon after birth in hypoxemic premature 

infants were associated with significant decreases in their oxidative stress after 

24 hr as assessed by plasma MDA. 105


NEONATAL NECROTIZING ENTEROCOLITIS 

Neonatal necrotizing enterocolitis (NEC) is the most common life-threatening 

disease of the gastrointestinal tract in the neonatal period; it primarily affects 

premature infants. 106 NEC is characterized by various degrees of mucosal or 

transmural necrosis of the intestine. Its causes remain unclear but are most likely 

multifactorial. Intestinal ischemia and colonization of the intestinal lumen by 

several viral and bacterial organisms were hypothesized to cause NEC. 107 Immunologic 

immaturity of the preterm gut 108 and neonatal hypoxia were also included 

as causative factors. Upon reperfusion and reoxygenation of the damaged intestine, 

a flood of ROS is generated by the xanthine oxidase system. This burst of 

ROS can cause severe tissue damage and may be the final pathway for tissue 

injury of the gut mucosa noted in NEC. 71 

ROS can alter inflammation in the gut and lead to activation of platelet 

activating factor (PAF), an endogenous phospholipid mediator 109 that results in 

increased coagulation and subsequent microthrombus formation in small vessels. 

A model of acute inflammatory intestinal injury induced by PAF was significantly 

attenuated by administration of allopurinol (a xanthine inhibitor). 110 

PERIVENTRICULAR LEUKOMALACIA 

Periventricular leukomalacia (PVL) in premature infants is a distinctive lesion of 

cerebral white matter (Figure 5.5) associated with severe adverse neurologic 

outcomes. The pathogenesis of cerebral white matter injury in premature infants 

FIGURE 5.5 Coronal sections of brain with periventricular leukomalacia.


is not entirely clear, although ischemia-reperfusion and infection-inflammation 

appear important. 70 The brain is at special risk due to its high concentration of 

PUFAs and deficiency of SOD and GPx. 111 Immature oligodendrocytes are more 

prone to oxidative stress than mature ones. 112 These immature forms, the so-called 

preoligodendrocytes, have been shown recently to account for 90% of the total 

population of oligodendrocytes in cerebral white matter of infants under the 

gestational age of 31 weeks 113 who represent a high risk group for the development 

of PVL. 114 

Houdou et al. also reported that CAT-positive glia did not appear in the deep 

white matter before 31 to 32 weeks of gestation. 115 These very premature infants 

are most at risk for white matter injury. 116 In two model systems of free radical 

accumulation, the early differentiating oligodendrocyte was shown to be exquisitely 

vulnerable to free radical attack. 113,117 

Direct support for an association between products of oxidation and PVL in 

premature infants is limited. However, in a recent study, premature infants with 

subsequent evidence of PVL on magnetic resonance imaging at term had higher 

levels of cerebrospinal fluid protein carbonyls (markers of oxidized protein) than 

healthy premature or term infants. 116 

RETINOPATHY OF PREMATURITY 

Retinopathy of prematurity (ROP) is a vasoproliferative retinal disorder of premature 

infants. Its basic pathogenesis is not fully understood but exposure to the 

extrauterine environment including necessarily high inspired oxygen concentrations 

produces cellular damage mediated by ROS. Hyperoxygenation favors peroxidation 

of vasoactive isoprostanes, resulting in vasoconstriction and vascular 

cytotoxicity leading to ischemia, which predisposes to the development of vasoproliferative 

retinopathy. 118 Severe ROP can lead to lifelong visual impairment 

or blindness. 119 

Early animal models of ROP first suggested that oxygen was involved in the 

normal developing retinal vasculature, probably via a putative angiogenic factor 120 

that was subsequently identified as vascular endothelial growth factor (VEGF). 121 

VEGF plays an important role in endothelial cell proliferation, migration, and 

blood vessel formation 122,123 and in the development of ROP. 124 

Several studies have shown that greater variability of transcutaneous oxygen 

during the first 2 weeks of life is associated with the development of severe 

ROP. 125,126 The so-called STOP-ROP (supplemental therapeutic oxygen for prethreshold 

retinopathy of prematurity) trial enrolled about 600 premature infants 

with confirmed threshold ROP in at least one eye. The risks of adverse pulmonary 

events including BPD increased with the use of supplemental oxygen at pulse 

oximetry saturation of 96 to 99% compared with pulse oximetry targeting 89 to 

94%. However, this therapy had no significant effect on the progression of ROP. 127 

Supplementation of antioxidants has been reported to protect against ROP. A 

meta-analysis evaluated data from six randomized clinical trials of vitamin E 

prophylaxis (15 to 100 mg/kg/day from day 1 until discharge) that included a


total of 704 premature infants in the vitamin E prophylaxis groups and 714 control 

infants. The overall incidence of any stage ROP was similar between the groups, 

but incidence of severe (stage +3) ROP was lower in the vitamin E-treated group 

(2.4% in the vitamin E group versus 5.3% of controls). The authors concluded 

that the role of the vitamin E antioxidant in reducing severe ROP must be reevaluated. 

128 

HUMAN MILK AND ANTIOXIDATIVE PROTECTION 

Human milk (HM) is considered the ideal food for healthy infants 129 and is known 

to contain various bioactive substances, some of which are reported to be antioxidants. 

130 HM contains many antioxidants such as enzymes (CAT, GPx, SOD), 

vitamins (A, C, E), binding proteins such as lactoferrin, and constituents of 

antioxidative enzymes (Cu, Zn). 131–133 In contrast, antioxidative enzymes are 

absent from infant formula. 134 Most infant formula has higher amounts of vitamins 

added than are present in HM to make up for the reduced bioavailability. Thus, 

the overall antioxidant capacity of HM versus infant formula is difficult to assess, 

although assessment would probably favor HM. 14 

We previously reported in vitro results showing that HM alleviated H 2O 2induced 

oxidative damage in intestinal epithelial cell lines, whereas bovine milk 

or infant formula did not. 135 Confluent intestinal epithelial (IEC-6) cells were 

preincubated with defatted HM, bovine milk, or three artificial milks for 24 hr 

followed by H 2O 2 challenge. HM-treated cells showed the highest survival rates 

(50%) compared with bovine milk-treated (6%) or infant formula-treated (13 to 

16%) cells (Figure 5.6). 135 Buescher and Mcllherhan 131 reported that human 

colostrum manifested antioxidant properties, proving capable of spontaneous 

reduction of cytochrome C, depletion of polymorphonuclear leukocyte-produced 

H 2O 2, and protection of epithelial cells from polymorphonuclear leukocyte-mediated 

detachment. 

Several clinical studies demonstrated antioxidative properties of HM. HMfed 

infants had higher plasma trapping ability (a measure of resistance to oxidative 

stress in vitro) than did control infants fed formula. 136 We compared the oxidative 

stress levels in 41 healthy 1-month-old infants and 29 premature infants by 

measuring urinary 8-hydroxy-2-deoxyguanosine (8-OHdG — a marker of oxidative 

DNA damage). In the 1-month-old group, urinary 8-OHdG excretions of the 

breast-fed infants were significantly lower than those of the artificial formula-fed 

infants (Figure 5.7). 137 In the premature group, urinary 8-OHdG excretions of the 

breast-fed infants at 14 and 28 days of age were significantly lower than those 

of the formula-fed infants (Figure 5.8). 138 These data indicate that HM provides 

more antioxidant properties than infant formula during early infancy. This may 

be due to the presence of antioxidants in HM that may exhibit antioxidant effects 

in the gut and may pass through the relatively porous neonatal intestine early in 

infancy. 131,132 In fact, feeding with HM has been associated with a lower incidence 

of a variety of illnesses including NEC, 139 respiratory disease, 140 and ROP 141 in 

premature infants.


Cell viability (% of control) 

60 

50 

40 

30 

20 

10 

0 

No pretreatment 

Human 

colostrum 

Infant formulas 

(a) (b) (c) 

Cow’s 

milk 

FIGURE 5.6 Antioxidative properties of human colostrum, bovine milk, and artificial 

formulas and H 2O 2-induced oxidative damage in IEC-6 cells. Survival cell rates are 

expressed as percentages of control (non-H 2O 2-challenged) cells. Values are means + SD. 

* p formula 

group received 50 to 90% of their intake as breast milk. Formula>breast group received 

50 to 90% of their intake as formula. Formula-fed group received 90% of their intake as 

formula. Values = means + SD. * p


Urinary 8-OHdG 

excretion (ng/mg . Cr) 

5 

4 

3 

2 

1 

0 

∗ 

Day 14 

FIGURE 5.8 Change in urinary 8-hydroxydeoxyguanosine (8-OHdG) excretion at 14 and 

28 days of age in breast-fed and formula-fed very low birthweight infants. Values = means 

+ SD. *p


way anticipates future policies in this area. BK is the recipient of a Freedom to 

Discover Award of the Bristol Myers Squibb Foundation, New York, NY, U.S.A. 

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6 

CONTENTS 

Maternal Obesity, 

Glucose Intolerance, 

and Inflammation in 

Pregnancy 

Janet C. King 

Abstract ...............................................................................................................93 

Introduction .........................................................................................................94 

Obesity, Inflammation, and Insulin Resistance ..................................................94 

Pregnancy: An Inflammatory, Insulin-Resistant State........................................96 

Maternal Obesity and Inflammation...................................................................98 

Maternal Inflammation, Insulin Resistance, and Fetal Growth .......................100 

Interventions to Reduce Maternal Inflammation and Insulin Resistance ........101 

Conclusions .......................................................................................................103 

Acknowledgments .............................................................................................103 

References .........................................................................................................103 

ABSTRACT 

The prevalence of obesity among pregnant women is at an all-time high. Maternal 

obesity increases the risks of mortality and morbidity in the mother and baby. 

The risk of gestational diabetes, one of the most prevalent metabolic complications 

in pregnancy, is significantly greater among obese women. Emerging 

research suggests an association of obesity, inflammation, and insulin resistance 

in non-pregnant and pregnant individuals. Cytokines secreted by the placenta, 

such as tumor necrosis factor-α and leptin, may mediate that link in pregnancy. 

Studies of maternal body mass index (BMI), insulin resistance, and circulating 

levels of cytokines, i.e., tumor necrosis factor-α and C-reactive protein, show that 

maternal obesity is associated with increased levels of inflammatory markers and 

that elevated levels of these markers are linked to glucose intolerance in women. 

These metabolic adjustments may exacerbate fetal overgrowth and excessive 

93


deposition of fat stores. Currently, obese pregnant women do not receive any 

guidance for reducing the risk of developing a pro-inflammatory, insulin-resistant 

state. Preliminary studies suggest that moderate physical activity and consuming 

a diet high in fiber and a higher proportion of polyunsaturated fatty acids may 

be beneficial. 

INTRODUCTION 

The prevalence of obesity among women of reproductive age has reached an alltime 

high. National survey data show that the number of women with body mass 

indexes (BMIs) greater than 30 averaged 31% in white women, 40% in Hispanic 

women, and 51% in black women in 1999 and 2000. 1 Obese women encounter 

more health problems during pregnancy. 1 Common disorders include pregnancyinduced 

hypertension, pre-eclampsia, large-for-gestational age (LGA) babies, 

need for cesarean or assisted deliveries, and post-delivery infections. The risk of 

gestational diabetes mellitus (GDM) is about three- to four-fold higher among 

obese compared to lean women. 2,3 The incidence of impaired glucose tolerance 

(IGT), i.e., one abnormal blood glucose value after an oral glucose load, is likely 

to be more frequent than GDM in obese women. 

The prevalence of GDM varies widely within and between populations. 

Although the U.S. national average is about 4%, 4 the prevalence is as high as 

16% in some high-risk populations. As with type 2 diabetes, the prevalence varies 

by race, ethnicity, and BMI. For example, in a study of 28,330 women from the 

Northern California Kaiser Permanente Medical Care Program, 7.4% of the Asian 

women, 5.6% of the Hispanic women, 4% of the African-American women, and 

3.9% of the white women developed GDM. 5 The rates among American Indian 

mothers were considerably higher: 15.1% among Zuni women and 10.4% among 

Navajo women. 4 Some of the differences due to race or ethnicity reflect the higher 

prevalence of obesity in certain populations. A study of 552 African-American 

women and 653 Latina women in Detroit showed that very obese Latina women 

(BMIs >35) demonstrated a 6.5-fold increased risk for developing GDM compared 

to normal weight Latina women; very obese African-American women had 

nearly a four-fold increased risk compared to normal weight women. 6 Although 

the risk of GDM increased with maternal body weights in both ethnic groups, 

the increase among Latina women was about 2.5 times greater than that in 

African-American women. 

OBESITY, INFLAMMATION, AND INSULIN RESISTANCE 

Obesity in non-pregnant adults is associated with subclinical inflammation and 

insulin resistance. 7 The inflammatory and insulin-resistant states arise from 

changes in cellular and molecular functions and metabolism when adipocytes 

become enlarged in obese individuals. Perlipin, a phosphoprotein on the surfaces 

of triglyceride droplets that acts as a gatekeeper preventing lipases from

Maternal Obesity, Glucose Intolerance, and Inflammation in Pregnancy 95 

Enlarged 

Adipocytes 

Macrophage 

Infiltration 

↑ TNF-α 

↑ IL-6 

↑ CRP 

INSULIN RESISTANCE 

↑ Lipolysis 

↑ Free Fatty Acids 

FIGURE 6.1 Relationship of obesity, inflammation, and insulin resistance. Enlarged adipocytes 

in the adipose tissues of obese individuals cause enhanced production of proinflammatory 

cytokines that increase rates of lipolysis, circulating levels of free fatty acids, 

and subsequent tissue insulin resistance. 

hydrolyzing triglycerides and releasing free fatty acids, declines when adipocytes 

become enlarged, leading to an increased release of free fatty acids. 7 In turn, free 

fatty acids promote insulin resistance by stimulating the phosphorylation of 

insulin-receptor substrate-1 (IRS-1) on its serine residues instead of the usual 

phosphorylation of tyrosine residues. 8 Once serine is phosphorylated, IRS-1 

becomes a poor substrate for the insulin receptor and insulin sensitivity declines. 

Cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) 

also stimulate IRS-1 phosphorylation on the serine residues. 

Obesity appears to predispose individuals to a pro-inflammatory state because 

enlarged fat stores promote the invasion of macrophages into adipose tissue to 

scavenge moribund adipocytes that tend to increase with obesity 7 (Figure 6.1). 

Thus, obese individuals often have increased circulating levels of cytokines such 

as TNF-α and IL-6. The cause of the increased number of moribund adipocytes 

in obese individuals is unknown, but one hypothesis is that clusters of adipocytes 

distant from capillary networks experience hypoxia before angiogenesis occurs. 9 

Cytokine secretion by macrophages also appears to be potently stimulated by 

acute-phase serum amyloid A; the decline in circulating levels of this protein 

after weight loss by obese individuals is associated with a reduction in circulating 

cytokine levels. 10 

The mild inflammatory state resulting from the increased secretion of cytokines 

by enlarged adipocytes contributes to the metabolic adjustments and insulin 

resistance associated with obesity. Some cytokines are thought to reduce adiponectin 

expression, 7 and serum adiponectin levels tend to fall with obesity. 11 

Adiponectin is a potent inhibitor of TNF-α-induced monocyte adhesion, which 

may explain in part the link between obesity and cardiovascular disease. 

TNF-α also increases adipocyte lipolysis, possibly by reducing perlipin 7 and 

thereby promoting the release of free fatty acids into circulation and reducing 

insulin sensitivity. IL-6 expression is also increased in the adipocytes of obese


individuals with higher levels in visceral than in peripheral adipose tissue. 7 The 

elevated plasma IL-6 levels in obese individuals are also associated with an 

increase in lipolysis. Thus, both TNF-α and IL-6 enhance the breakdown of fats 

to free fatty acids in adipose tissue, increasing their delivery to liver and muscle 

and subsequently causing insulin resistance. 

Endogenous overproduction of cortisol or exogenous administration of cortisteroids 

generally causes weight gain with an increase in visceral fat stores 

compared with peripheral stores. However, obese people usually do not have 

higher levels of cortisol. 7 The activity of 11β-hydroxysteroid dehydrogenase type 

1, which converts inactive cortisol metabolites into active cortisol, is elevated in 

the adipose tissues of obese people. Higher rates of cortisol production in adipose 

tissue may contribute to hyperphagia, increased cytokine expression, gain in 

visceral adipose tissue, hyperlipidemia, and insulin resistance in obese individuals 

without any increases in circulating cortisol concentrations. 

Clearly, adipocytes are not merely storage reservoirs for fat, but they are also 

endocrine organs with multiple functions. Their metabolic function changes as 

they enlarge with increasing obesity. Enlarged adipocytes recruit macrophages, 

promote inflammation, and enhance the secretion of a variety of metabolites that 

predispose toward insulin resistance. An active area of research is identification 

of therapies that alter adipose tissue metabolic pathways leading to inflammation 

and insulin resistance. 

PREGNANCY: AN INFLAMMATORY, INSULIN-RESISTANT STATE 

Profound metabolic adjustments occur during pregnancy to assure an adequate 

nutrient supply is available to support fetal growth. Since glucose is the preferred 

fuel of the fetus, maternal metabolism is shifted toward a hyperglycemic state. 

This ensures facilitated glucose diffusion from maternal circulation across the 

placenta to the fetus. Maternal hyperglycemia is created by establishing an insulin-resistant 

state. Maternal insulin resistance increases throughout gestation, 

reaching a peak in late gestation when fetal fuel demands are the highest. 12 The 

rise in insulin resistance is ascribed to alterations in maternal cortisol levels and 

placental hormones (human placental lactogen, progesterone, and estrogen). 12 

However, the changes in insulin resistance have never been correlated with these 

hormonal changes in a prospective, longitudinal study. 13 

The recent evidence that adipokines such as TNF-α and leptin affect insulin 

sensitivity in non-pregnant individuals has led investigators to propose that similar 

mechanisms may occur in pregnancy. Pro-inflammatory cytokines may influence 

insulin metabolism in pregnant as well as non-pregnant women for several reasons. 

First, pregnant women with adequate food supplies gain fat during the first 

two trimesters. 1 The amounts gained vary from 0 to 10 kg and the average is 

about 3.5 kg. Expansion of the fat tissue and enlarged adipocytes may increase 

adipose tissue cytokine secretion and subsequent insulin resistance in pregnant 

women similar to that seen in non-pregnant individuals. Second, the placenta


expresses all known cytokines including TNF-α, IL-6, IL-10, leptin, resistin, and 

PAI-1. 14 

The physiological role of these placental cytokines is uncertain, but many of 

the same cytokines are produced by the placenta as are secreted by adipose tissue. 

Possibly, the high placental cytokine secretion assists in creating the maternal 

insulin-resistant state. Since maternal fat gain is very limited or non-existent in 

pregnant women with limited energy intakes, the placenta is likely to be more 

important for creating a maternal insulin-resistant state than is an increase in 

maternal adipose tissue. 

To test the hypothesis that placental cytokines play a role in modifying insulin 

sensitivity in pregnancy, Kirwan and co-workers measured circulating hormones, 

cytokines, and insulin resistance in 15 women (5 with GDM and 10 with normal 

glycemia) before pregnancy, at 12 to 14 weeks of gestation, and at 34 to 36 weeks 

of gestation. 13 Changes in insulin sensitivity were compared to placental hormones, 

cortisol, leptin, and TNF-α. TNF-α was more strongly associated with 

insulin sensitivity than any other hormone or cytokine measured; higher TNF-α 

levels were associated with lower insulin sensitivity (r = –0.69, p


those observed in late pregnancy. 16 This confirmed that the levels of free fatty 

acids play an important role in regulating insulin sensitivity in pregnancy. Recent 

research suggests that the circulating levels of cytokines secreted by the placenta 

may mediate shifts in free fatty acid concentrations and subsequent insulin resistance. 

MATERNAL OBESITY AND INFLAMMATION 

Studies of inflammation and insulin resistance in pregnancy were performed in 

non-obese women. 13,17 Since obesity precipitates inflammatory responses, excessive 

free fatty acid release, and subsequent insulin-resistant states in non-pregnant 

individuals, it is reasonable to assume that inflammatory responses and insulin 

resistance would be enhanced in obese compared to lean pregnant women. Comparisons 

of metabolic adjustments in lean and obese pregnant women are limited, 

but the few studies done show that obese women rely more on fat oxidation as 

a source of energy in late pregnancy than do lean women. 18,19 

The increase in fat oxidation among the obese women was significantly 

correlated with serum leptin concentrations (r = 0.76, p


concentrations following oral glucose tolerance tests. 23 Insulin sensitivity declined 

by 34% in the non-obese group from 12 to 34 weeks and by 60% in the obese 

group; the change in the two groups did not achieve statistical significance (p = 

0.09). Insulin sensitivity did not differ between the two groups at 12 weeks of 

gestation, but it was significantly lower in the obese than in the non-obese at 34 

weeks (p


MATERNAL INFLAMMATION, INSULIN RESISTANCE, AND 

FETAL GROWTH 

Obese women tend to have big babies irrespective of the amount of weight they 

gain. 26 Also, maternal glucose metabolism and adiposity are highly correlated 

with fetal growth and body composition. 21,27 Although gestational age at birth is 

the strongest predictor of both birth weight and infant fat-free mass, maternal 

pregravid BMI is the strongest predictor of infant fat mass (r 2 = 0.066), explaining 

about 7% of the variance in body fat of newborns. 

Maternal diabetes is a strong predictor of fetal overgrowth, with diabetic 

women facing a three-fold higher risk of having an LGA baby than obese women. 

Four times as many LGA infants are born to obese women than to diabetic women 

because there are many more obese women than diabetic women. Also, diabetic 

women usually are carefully monitored by their health care providers during 

pregnancy whereas no special care is provided to obese women. The additional 

care given to diabetic women undoubtedly reduces the number of LGA babies 

born to them. Recent studies from both North America and Europe report 

increases in mean birth weight over the past 30 years. 21 This rise in birth weight 

is likely related to the increasing incidence of maternal obesity. In fact, Catalano 

and his co-workers in Cleveland, Ohio found that a mean increase in birth weight 

of 116 g over the past 30 years was more strongly correlated with maternal weight 

at delivery than any other maternal characteristic. 

Is this rise in birth weight linked to an increased maternal subclinical inflammation 

and insulin resistance associated with obesity? Preliminary data suggest 

that it may be. 28 For example, Radaelli and co-workers 29 measured circulating 

maternal and fetal cytokines and growth factors in three mother–infant cohorts 

divided into tertiles according to neonatal body fat. The only fetal factor associated 

with neonatal body fat was leptin (p


INTERVENTIONS TO REDUCE MATERNAL INFLAMMATION AND 

INSULIN RESISTANCE 

Studies in non-pregnant obese individuals provided evidence that changes in 

lifestyle (weight reduction, changes in dietary fat and fiber, and increased physical 

activity) reduced the risks of type 2 diabetes mellitus in obese individuals. 31 

Weight loss is a very effective way to reduce circulating cytokine levels and 

fasting insulin concentrations in obese women 32 but weight loss is not indicated 

for pregnant women. However, modest reductions in the total amounts gained by 

obese women are appropriate. The Institute of Medicine recommends that obese 

women gain at least 15 lb during gestation; normal weight women are advised 

to gain 25 to 35 lb. 33 

Physical activity is an effective intervention for reducing the risk of type 2 

diabetes and associated metabolic anomalies such as insulin resistance, oxidative 

stress, and dyslipidemia. 34 Physical activity activates the AMP-activated protein 

kinase (AMPK) enzyme, which increases glucose transport into the muscle, 

enhances fat oxidation, and reduces insulin resistance. 7 . Exercise, even intermittently, 

reduces the risk of GDM among obese women with BMIs >33 by nearly 

two-fold. 35 Women who exercise throughout pregnancy (i.e., perform endurance 

exercises ≥4 times/week) gain significantly less fat and had significantly lower 

increases in TNF-α and leptin during gestation. 36 The changes in leptin, but not 

TNF-α, were correlated with reduced fat mass in physically active women. 

Possibly, the differences in TNF-α levels reflect the exercise-induced reductions 

in insulin resistance whereas the leptin changes are more closely linked to fat 

accretion. Nevertheless, moderate physical activity during pregnancy may be an 

effective way to reduce subclinical inflammation and insulin resistance during 

pregnancy. 

Changes in the types of dietary fat and carbohydrates consumed may be other 

ways to reduce maternal inflammation and insulin resistance. Decreases in total 

fat and saturated fat intakes and increases in dietary fiber are recommended for 

reducing the risk of type 2 diabetes mellitus in non-pregnant adults. 37 Results 

from the limited number of studies of pregnant women suggest that similar 

changes in dietary fats and carbohydrates also effectively reduced the risks of 

glucose intolerance. Clapp randomized 12 pregnant women to a low or high 

glycemic index diet prior to conception. 38 The amounts of carbohydrate consumed 

were similar in the two groups: 56% of the total energy. 

During pregnancy, the women on the low glycemic diet showed no significant 

changes in their glycemic responses to mixed meals whereas the women on the 

high glycemic diet experienced 190% increases in their responses compared to 

pre-pregnancy values. These findings are similar to those reported by Fraser et 

al. 39 who showed that a high fiber diet reduced the post-prandial response to a 

meal in comparison to low fiber intake. Bronstein and co-workers have also shown 

that reducing the glycemic index of a test meal lowered post-prandial glucose 

and insulin responses in both lean and obese women studied in the third trimester 40 

(Figure 6.3). These findings suggest that the maternal insulin-resistant state and


mg/mLxhrs 

uU/mLxhrs 

250 

200 

150 

100 

50 

0 

900 

800 

700 

600 

500 

400 

300 

200 

100 

0 

Insulin Area Under Curve 

High GL Lower GL 

Glucose Area Under Curve 

High GL Lower GL 

Obese 

Non-obese 

FIGURE 6.3 Serum insulin and glucose responses to lower glycemic test meals in lean 

and obese pregnant women. The areas under the curve for serum insulin and glucose were 

measured over a 2-hour period in 6 lean and 8 obese pregnant women studied at 32 to 36 

weeks of gestation. 40 

hyperglycemic responses to meals reflect the intake of a Westernized, low fiber, 

high glycemic diet rather than a typical metabolic response to pregnancy. 

Studies of non-pregnant, obese individuals also suggest that increasing the 

ratio of polyunsaturated to saturated fats in the diet may reduce the risk of 

developing metabolic syndrome and its complications as early as adolescence. 41 

Similar findings have been reported for pregnant women. In a study of 171 

pregnant Chinese women with or without impaired glucose tolerance, the type 

of dietary fat predicted impaired glucose tolerance and GDM. 42 In a logistic 

regression analysis, increased body weight, decreased polyunsaturated fat intake, 

and a low dietary polyunsaturated-to-saturated fat ratio independently predicted 

glucose intolerance. Bo and co-workers also found that glucose intolerance in 

pregnant women without conventional risk factors (i.e., family history, age, and


BMI) was related to the percent of saturated and polyunsaturated fats in the diet 

with high intakes of saturated fat increasing the risk and high intakes of polyunsaturated 

fat decreasing the risk. 43 

The conventional dietary treatment of women with GDM is to reduce the 

amount of dietary carbohydrate and increase slightly the amount of fat. The 

preliminary data reviewed here suggest that it is more important to consider the 

types of carbohydrates and fats rather than the amounts. Increasing dietary fiber 

(or lowering the glycemic index) and the proportion of polyunsaturated fatty acids 

may be effective interventions for reducing inflammation and insulin resistance 

in pregnancy. 

CONCLUSIONS 

Placental cytokine secretion induces a pro-inflammatory state during pregnancy. 

This metabolic state is exacerbated by additional cytokines secreted by enlarged 

adipocytes in obese pregnant women. The inflammatory state leads to an increase 

in circulating free fatty acids and an insulin-resistant state. This pro-inflammatory, 

insulin-resistant state in a mother may affect placental function and fetal growth 

and development. Accumulating evidence suggests that the fetal fuel supply is 

enhanced, leading to accelerated growth and excessive fetal fat stores. Currently, 

obese pregnant women are not given any specific advice for reducing the proinflammatory 

response and insulin resistance. However, studies in both nonpregnant 

and pregnant adults suggest that moderate physical activity and 

increased intakes of dietary fiber and the proportion of polyunsaturated fatty acids 

attenuate these metabolic abnormalities and thereby potentially improve pregnancy 

outcomes. 


The author acknowledges the contributions of her research collaborators to the 

original, unpublished research findings included in this paper: Jessica DeHaene, 

Dina El Kady, James L. Graham, Peter J. Havel, Liza Kunz, Meredith Milet, 

Ratna Mukherjea, Kimber L. Stanhope, and Leslie R. Woodhouse. 

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972.

7 

CONTENTS 

Obesity, Nutrigenomics, 

Metabolic Syndrome, 

and Type 2 Diabetes 

David Heber 

Introduction .......................................................................................................107 

Obesity Epidemic and Its Solutions .................................................................108 

Type 2 Diabetes Mellitus and Obesity: “Diabesity”........................................111 

Metabolic Syndrome: Pre-Diabetes or Cardiometabolic Risk? .......................111 

Nutrigenetics of Type 2 Diabetes Mellitus.......................................................113 

Beta Cell Failure and Type 2 Diabetes Mellitus..............................................115 

Adipokines ........................................................................................................116 

Conclusion.........................................................................................................118 

References .........................................................................................................118 

INTRODUCTION 

The traditional medical paradigm for understanding diabetes mellitus has undergone 

a major shift in the past two decades with the discovery of the many 

immunological functions of fat cells, especially those located in the mesenteric 

or visceral fat of the abdomen. This shift in thinking occurred in the molecular–genetic 

era of the last decade, beginning with the discovery of leptin. 

Once it was realized that leptin is also a cytokine that stimulates angiogenesis, 

the many connections between obesity, inflammation, and diabetes began to 

emerge. A large number of cytokines and chemokines originating in fat cells were 

subsequently discovered. Recently, our group has demonstrated that a small but 

significant 5% weight loss could lead to a much larger decrease in the levels of 

circulating C-reactive protein, a biomarker of inflammation. This connection of 

inflammation and obesity, leading in genetically susceptible individuals to diabetes 

mellitus type 2, provides a unifying pathophysiological mechanism for 

many of the co-morbid diseases associated with diabetes and the metabolic 

syndrome including cardiovascular disease, renal disease, liver disease, and 

hypertension. 

107


OBESITY EPIDEMIC AND ITS SOLUTIONS 

Obesity is defined as excess body fat, but the difficulties inherent in measuring 

body fat in tens of thousands of individuals have resulted in the substitution of 

a surrogate measure called body mass index (BMI), defined as weight in kilograms 

over height in meters squared. This is a regression equation that approximates 

excess body fat in large populations, but can be significantly erroneous in 

individuals such as athletes, whose excess weight is due to muscle, or in young 

sedentary women consuming inadequate dietary protein who may have low body 

weights with abnormally high percentages of body fat. 

Practical methods such as bioelectrical impedance can be used to assess body 

fat in individuals in clinical settings, but the BMI has proven invaluable in 

population studies. Overweight, defined as a BMI of at least 25 but less than 30 

kg/m 2 , and obesity (used here to denote greater overweight), defined as a BMI 

of 30 kg/m 2 or greater, are major contributors to morbidity and mortality in the 

United States today. The risk for some of our most devastating diseases, especially 

cardiovascular disease and diabetes mellitus, is significantly correlated with an 

individual’s BMI 1 and a huge portion of the country's healthcare resources are 

used to treat the consequences of overweight and obesity. The United States is 

in the midst of an explosive epidemic of obesity. Although the early maps from 

1985 to 1990 present all states in shades of light blue, indicating obesity prevalences 

of less than 15%, the latest maps are almost entirely orange (20 to 24% 

prevalence), with an ominous streak of red (25% or more) arcing from Texas to 

Michigan through Appalachia. 2 (Maps are available at http://www.cdc.gov/nccdphp/dnpa/obesity/trend/maps/.) 

Weight gain is primarily a matter of energy imbalance — energy intake that 

is greater than energy output. This imbalance is influenced by genetic, metabolic, 

behavioral, and environmental factors. Only the latter two factors could have 

changed enough in the last 20 years to cause the obesity epidemic, resulting in 

both an increase in average energy intake and a decrease in average energy 

expenditure. Energy intake has increased in the United States due to a greater 

availability of highly palatable and energy-dense foods, larger portions becoming 

the norm, more food consumed outside the home (where less heed is paid to 

nutrition), and greater consumption of high-sugar beverages and high-fat processed 

and fast foods. 

Over the past 20 years, nutrition researchers and food scientists over-emphasized 

reducing fat consumption with less regard for total calories, resulting in an 

ineffective low-fat food campaign while the incidence of obesity continued to 

increase. A significant decrease in energy output has been associated with 

increased hours of television viewing, increased numbers of individuals in sedentary 

jobs, and the increased prevalence of labor-saving devices such as elevators, 

automobiles, and remote controls. Some experts hold that the current obesity 

epidemic is caused by relatively small but chronic energy imbalances. Because 

a pound of body fat represents 3500 stored calories, a chronic energy imbalance 

— intake over output — of only 100 calories per day will cause a weight gain

Obesity, Nutrigenomics, Metabolic Syndrome, and Type 2 Diabetes 109 

TABLE 7.1 

Patient Assessment: Factors to Consider in Developing Weight Loss 

Strategies 

Factor Guidelines 

Body mass BMI 25.0 to 29.9 kg/m 

index (BMI) 

2 (overweight) 

BMI ≥30 kg/m2 (obese) 

Waist 

Men: >102 cm (40 inches) 

circumference Women: >88 cm (35 inches) 

Risk status High: 

Presence of coronary heart disease, atherosclerosis, type 2 diabetes, sleep 

apnea, OR any three of the following: 

Cigarette smoking 

Hypertension (systolic blood pressure ≥140 mm Hg or diastolic blood pressure 

≥90 mm Hg) 

High LDL cholesterol (≥160 mg/dL) 

Low HDL cholesterol (200 mg/dL) 

Patient 

Reasons and motivation for weight reduction 

motivation History of successful and unsuccessful weight loss attempts 

Support (family, friends, co-workers) 

Patient's understanding of causes and health risks of obesity 

Attitude toward and capacity for physical activity 

Time available for weight loss intervention 

Financial considerations 

Adapted from NIH/NHLBI clinical guidelines on overweight and obesity. Body mass index (kg/m 2 ) 

can be calculated from pounds and inches using the formula: BMI (kg/m 2 ) = [weight 

(pounds)/height (inches) 2 ] × 704.5. An interactive BMI calculator and BMI chart are included in 

the NIH/NHLBI report. 

of 10 pounds a year. The only way to restore energy balance is to reduce energy 

intake and/or increase energy output. 

The assessment process, as described in the National Institutes of Health/ 

National Heart, Lung, and Blood Institute report titled Clinical Guidelines on the 

Identification, Evaluation, and Treatment of Overweight and Obesity in Adults, 

involves considering a patient's BMI, waist circumference, motivation to lose 

weight, and overall risk status (Table 7.1). 

Weight reduction has well documented health benefits and can usually be 

achieved with a loss of 1 to 2 pounds per week (0.5 to 1.0 pounds for lower


starting weights), requiring an energy deficit of 500 to 1000 calories per day for 

the obese or 300 to 500 calories per day for the overweight. Achieving this energy 

deficit requires a three-part strategy that includes dietary therapy, physical activity, 

and behavior therapy. 

Dietary therapy may be the most controversial of these elements. Patients 

tend to diet to achieve weight loss and then stop when their goals are reached. 

However, obesity is a chronic disease that cannot be cured and must be controlled 

through permanent lifestyle changes. Indications of what comprises successful 

diet strategies have been gleaned from the National Weight Control Registry 

(NWCR), which is following more than 4500 adults who were able to maintain 

weight losses of at least 30 pounds for at least a year. The typical participant 

(with an average weight loss of 60 pounds maintained for 5 years) made a 

permanent change to a low-calorie diet, what is called “chronic restrained eating.” 4 

There are many possible means to attaining a state of chronic restrained 

eating. Thus, it makes little difference which form of diet one chooses to follow, 

whether it involves restricted carbohydrates (e.g., Atkins), macronutrient balance 

(e.g., Zone), restricted fat (e.g., Ornish), or simply restricted portion sizes and 

calories (e.g., Weight Watchers); what matters primarily is whether one is able 

to adhere to the diet. 5 Once a weight goal is achieved, there is general agreement 

that an optimally healthy diet consisting of seven servings per day of colorful 

fruits and vegetables, whole grains, low fat protein sources, and limited amounts 

of refined carbohydrates should be consumed. Significant amounts of research 

also suggest that structured diet plans using meal replacements such as high 

protein shakes or frozen meals are helpful adjuncts to weight loss. The most 

important requirements for an effective diet are that it (1) establishes a calorie 

deficit, (2) is healthy, and (3) fits one’s lifestyle. 

The next component of a successful weight loss strategy is physical activity. 

In fact, a daily expenditure of 300 or more calories through physical activity may 

be the most important factor for maintaining weight loss according to the NWCR. 

To accomplish this, a formerly obese person would need to perform 60 to 90 

minutes per day of moderate-intensity physical activity, equivalent to walking at 

3.5 to 4.0 miles per hour. Lesser amounts are recommended to prevent excess 

weight gain or reduce the risk of chronic disease. 

A popular and simple program that started in Japan is the 10,000 steps per 

day idea, which provides a concrete goal. Since the goal can be worked on 

throughout the day, this strategy encourages cumulative episodes of physical 

activity. A goal counted in daily steps has been shown to induce more total 

walking than a goal measured in, say, hours of brisk walking. 6 

Abdominal obesity due to excess visceral fat is associated with an increased 

risk of developing cardiovascular disease. 1,7 Moreover, excess visceral fat is 

linked to an increased risk of metabolic syndrome, which includes a greater risk 

of developing type 2 diabetes mellitus 3 with its associated cardiometabolic 

disorders. 4


TYPE 2 DIABETES MELLITUS AND OBESITY: “DIABESITY” 

The focus of physicians treating diabetes has been strictly on glucose control for 

much of the last century. In fact, the term diabetes mellitus derives from the Latin 

meaning “sweet urine.” In Ayurvedic medicine, the term for diabetes 

(madhumeda) translates as “one whose urine attracts ants.” This glucocentric 

model of diabetes evolved from observations in the 1920s that surgical removal 

of the pancreas in a dog led to diabetes mellitus. Insulin injections could then 

restore glucose metabolism. 

In juvenile or type 1 diabetes mellitus patients with autoimmune destruction 

of the pancreas early in life, insulin treatment could delay or prevent many of 

the complications of this terrible disease such as blindness, renal failure, and 

need for limb amputations. However, this form of diabetes is much less common 

today, accounting for less than 5% of all diabetes cases. Today, over 95% of all 

diabetes is type 2 and it occurs in children and adolescents as well as adults. In 

an individual with a BMI of 30, the risk of diabetes type 2 is increased 60- to 

80-fold in comparison to lean individuals. In contrast, the risk for heart disease 

is only 4- to 6-fold increased at a BMI of 30 (see Figure 7.1). The association 

of diabetes type 2 with obesity goes beyond typical risk factors and justifies 

naming the disease diabesity. While 10% of type 2 diabetes patients are said to 

be lean, this assessment is based on weight and not body composition; many of 

these individuals may have excess abdominal fat. 

As type 2 diabetes mellitus gained recognition in the 1970s, its etiology 

remained poorly understood. The idea that insulin, by controlling complications, 

was central to therapy was simply applied to type 2 diabetes mellitus as it had 

been to type 1, with the assumption that the results would be the same. However, 

insulin treatment failed to reduce cardiovascular mortality. 

The late onset of diabetes mellitus type 2, usually in the fourth to sixth decade 

of life, was attributed to aging of the beta cell within the pancreas which secretes 

insulin. The pathophysiologic progression of type 2 diabetes mellitus was first 

described by Drenick and Johnson. Over a 2- to 10-year period, their model 

predicted evolution from hyperinsulinemia with euglycemia to a condition of 

insulin deficiency and hyperglycemia as the beta cell was exhausted. Dr. Peter 

Butler recently described a likely cause of beta cell exhaustion by uncovering 

the role of an amyloid protein called insulin-associated polypeptide or IAPP. 

METABOLIC SYNDROME: PRE-DIABETES OR 

CARDIOMETABOLIC RISK? 

A particular cluster of risk factors that seems especially coherent and predictive 

of atherosclerotic cardiovascular disease (ASCVD) has been called the metabolic 

syndrome. It comprises abdominal obesity, atherogenic dyslipidemia, hypertension, 

and elevated plasma glucose, along with the pro-thrombotic and pro-inflammatory 

states. Identification of this syndrome has proven useful in unifying


Relative Risk 

(a) 

Relative Risk 

(b) 

6 

5 

4 

3 

2 

1 

0 

6 

5 

4 

3 

2 

1 

0 

Women 

Type 2 diabetes 

Cholelithiasis 

Hypertension 

Coronary heart disease 

21 22 23 24 25 26 27 28 29 30 

Body-Mass Index 

Men 

Type 2 diabetes 

Cholelithiasis 

Hypertension 

Coronary heart disease 

21 22 23 24 25 26 27 28 29 30 

Body-Mass Index 

FIGURE 7.1 Relation of body mass index (BMI) and relative risk of diabetes, cholelithiasis, 

hypertension, and coronary heart disease. (Source: From Willett, W.C. et al. New 

Engl J Med 341, 427, 1999.) 

medical approaches to the previously disparate realms of diabetes and cardiovascular 

disease. 8 

The National Heart, Lung, and Blood Institute in conjunction with the American 

Heart Association recently published updated guidelines for the diagnosis 

of metabolic syndrome 9 (Table 7.2). Note that elevated low-density lipoprotein 

cholesterol (LDL-C) is not listed as a component; this is because LDL-C levels 

are not generally divergent from normal levels in metabolic syndrome. However, 

LDL particles are seen to be smaller and denser in the syndrome, so that small, 

dense LDL (sdLDL) particle size is an experimental observation in metabolic 

syndrome but is not practical to assess clinically. The unifying framework for the 

relationships of obesity, metabolic syndrome, type 2 diabetes, and cardiovascular 

disease is hypothesized to be the release of free fatty acids (FFAs) preferentially


TABLE 7.2 

Criteria for Diagnosing Metabolic Syndrome 

Three or more of the following characteristics define metabolic syndrome: 

1. Abdominal obesity: waist circumference >102 cm in men and >88 cm in women 

2. Hypertriglyceridemia: ≥150 mg/dL (1.69 mmol/L) 

3. Low high-density lipoprotein (HDL) cholesterol:


the past 100,000 years. It is likely that many forms of diabetes mellitus type 2 

are associated with numerous genetic polymorphisms concerning both inflammation 

and the adaptation to starvation. 

As of October 2005, 176 human obesity cases due to single-gene mutations 

in 11 different genes have been reported, 50 loci related to Mendelian syndromes 

relevant to human obesity have been mapped to a genomic region, and causal 

genes or strong candidates have been identified for most of these syndromes. 13 

There are 244 genes that, when mutated or expressed as transgenes in mice, 

result in phenotypes that affect body weight and adiposity. The number of studies 

reporting associations between DNA sequence variation in specific genes and 

obesity phenotypes has also increased considerably, with 426 findings of positive 

associations with 127 candidate genes. The nutrigenetics of diabetes mellitus type 

2 and obesity will most likely be closely related, if not identical, in many 

individuals with the difference being that environmental influences uncover 

underlying type 2 diabetes mellitus. 

The famous example of the Pima Indians in the Southwest United States 

points out the influence of environmental factors. Living only a few hundred 

miles apart, members of the same tribe were separated by the United States–Mexican 

border but were genetically similar. Members of the tribe living in the United 

States were forced onto a reservation in Arizona where they could not pursue 

their agricultural lifestyle. As a result, the age- and sex-adjusted prevalence of 

type 2 diabetes in the Mexican Pima Indians (6.9%) was less than one-fifth that 

in the U.S. Pima Indians (38%) and similar to that of non-Pima Mexicans (2.6%). 

The prevalence of obesity was similar in the Mexican Pima Indians (7% in men 

and 20% in women) and non-Pima Mexicans (9% in men and 27% in women) 

but was much lower than in the U.S. Pima Indians. 14 Other examples can be drawn 

both from immigrants to the U.S. or internationally in countries such as China, 

Japan, Korea, and Thailand where Western diets are rapidly replacing traditional 

Asian diets, leading to increases in obesity and diabetes. 

While the treatment strategies for this vast complex of medical problems 

traditionally includes treatment of the complications (hypertension, dyslipidemia, 

and type 2 diabetes), or management of the ensuing coronary heart disease risk, 

it is a challenge to incorporate prevention and reduction of the “hypertriglyceridemic 

waist” into standard medical practice. 

In patients who have already developed type 2 diabetes mellitus, a knowledge 

of proper blood glucose monitoring along with the ability to assess emotional 

status and garner adequate social support augment the efforts to increase physical 

activity and modify nutritional habits. Among the drugs used that can complement 

lifestyle change, metformin, which is off patent currently, is the drug of choice. 

Metformin blocks hepatic gluconeogenesis by an unknown mechanism, but has 

minimal effect on insulin resistance. It also reduces the prothrombotic state by 

an effect on plasminogen activator inhibitor type 1 (PAI-1), and its early use can 

prevent the progression from prediabetes to diabetes. Metformin’s effect on CVD 

risk is uncertain, although it does cause a modest weight loss.


Drugs aimed at increasing insulin secretion (e.g., the sulfonylureas) have 

shown no significant impacts on CVD risk and may even increase risk after a 

myocardial infarction (MI). 

Insulin limits markers of inflammation, reduces intimal medial thickness 

(IMT, a surrogate marker of CVD risk), and has been shown to lower risk after 

coronary artery bypass and to reduce post-MI mortality. On the whole, intensive 

treatment of hyperglycemia per se has been seen to have minimal effect on CVD 

risk. 15 

Besides insulin, metformin, and sulfonylureas, a new class of drug has 

emerged in the last decade, the thiazolidinediones (TZDs) or glitazones. These 

drugs are uniquely specific in reversing insulin resistance as agonists of the 

peroxisome proliferator-activated receptor gamma (PPAR-gamma). This is a 

nuclear receptor found in adipocytes and elsewhere that regulates the expression 

of genes involved in lipid and glucose metabolism, vascular function, thrombotic 

control, and inflammation. However, glitazones can also stimulate adipogenesis 

and so are used as second-line agents after metformin which encourages weight 

loss. 

Exenatide is a new injectable treatment developed after observation of the 

“incretin effect” in which insulin levels were seen to rise significantly more after 

oral glucose than after an IV injection of glucose due to the action of a thenunidentified 

gut peptide. 16 Exenatide is a synthetic version of a salivary peptide 

in the Gila monster, and it mimics an endogenous incretin, glucagon-like peptide 

1 in stimulating glucose-dependent insulin secretion, regulating gastric emptying, 

and inhibiting glucagon secretion, food intake, and acute plasma glucose. 

Exenatide is indicated as an adjunct to metformin and has been associated with 

weight loss as well. 

Another new injectable treatment is pramlintide, a more soluble analog of 

amylin, which is co-secreted by the pancreatic gamma cell with insulin. Amylin, 

and thus pramlintide, promotes satiety, inhibits glucagon release, and delays 

gastric emptying, all through an effect via the central nervous system. 16 

BETA CELL FAILURE AND TYPE 2 DIABETES MELLITUS 

Type 2 diabetes involves a progressive defect of insulin secretion that precedes 

the development of hyperglycemia. 17 This defect appears to be at least in part due 

to a deficit in beta cell mass. 18–20 Several therapeutic strategies now being proposed 

may reverse the defect in beta cell mass in people with type 2 diabetes, for 

example, glucagon-like peptide 1 or glucagon-like peptide 1–like surrogates. 21 

In humans there is a curvilinear relationship between the relative beta cell 

volume (and presumably the beta cell mass) and fasting blood glucose concentration. 

The present data reveal a narrow range of blood glucose over a wide range 

of fractional beta cell volume (up to ~10%) and then a much wider range of blood 

glucose values over a narrow range of volumes at low beta cell volumes, with 

the threshold set by the curve at ~1.1% defining that difference. These findings 

imply a much greater tolerance for variance in insulin sensitivity above this


threshold and that below-the-threshold variance in insulin sensitivity and functional 

defects in insulin secretion have a much greater impact on blood glucose. 

Autopsy studies involve some important limitations. The numbers of cases 

are frequently relatively small. The studies are inevitably cross-sectional and 

retrospective. Butler and others have presented data suggesting that the decline 

in beta cell mass in type 2 diabetes is caused by increased beta cell apoptosis. 19,22 

Therefore, these data suggest that inhibition of beta cell apoptosis to avoid a beta 

cell deficit may be effective to delay and/or avoid the onset of diabetes. Indeed, 

both metformin and TZDs have been reported to inhibit beta cell apoptosis in 

vitro 22,23 and delay onset of type 2 diabetes in clinical studies. 24,25 

The potential mechanisms underlying increased beta cell apoptosis in type 2 

diabetes include toxicity from islet amyloid polypeptide oligomer formation, free 

fatty acids (lipotoxicity), free oxygen radical toxicity, and, once hyperglycemia 

supervenes, glucose-induced apoptosis (glucotoxicity). 26,27 The steep increase in 

blood glucose concentration with beta cell deficiency is consistent with the well 

known deleterious effects of hyperglycemia per se on beta cell function. These 

include defective glucose sensing due to reduced glucokinase activity, 28 impaired 

glucose-induced insulin secretion due to increased uncoupling protein 2 activity, 29 

and depletion of immediately secretable insulin stores. 30 Also, hyperglycemia 

reduces insulin sensitivity, further compounding the effects of decreased insulin 

secretion. 31 While these observations suggest that relatively small increases in 

beta cell mass may have useful actions in restoring blood glucose control, it is 

likely that aggressive normalization of blood glucose concentrations is required 

to accompany any strategy to increase beta cell mass to overcome the deleterious 

effects of hyperglycemia as well as glucose-induced beta cell apoptosis. 

ADIPOKINES 

Adipose tissue secretes bioactive peptides, termed adipokines, which act locally 

and distally through autocrine, paracrine and endocrine effects. In obesity, 

increased production of most adipokines impacts on multiple functions such as 

appetite and energy balance, immunity, insulin sensitivity, angiogenesis, blood 

pressure, lipid metabolism and hemostasis, all of which are linked with cardiovascular 

disease. Enhanced activities of TNF and IL-6 are involved in the development 

of obesity-related insulin resistance. Angiotensinogen has been implicated 

in hypertension and PAI-1 in impaired fibrinolysis. 

Other adipokines like adiponectin and leptin, at least in physiological concentrations, 

are insulin sparing as they stimulate beta oxidation of fatty acids in 

skeletal muscle. The role of resistin is less understood. It is implicated in insulin 

resistance in rats, but probably not in humans. Reducing adipose tissue mass 

through weight loss in association with exercise can lower TNF-α and IL-6 levels 

and increase adiponectin concentrations, whereas drugs such as TZDs increase 

endogenous adiponectin production. In-depth understanding of the pathophysiology 

and molecular actions of adipokines may in the coming years lead to


effective therapeutic strategies designed to protect against atherosclerosis in obese 

patients. 

Obesity associated with unfavorable changes in adipokine expression such 

as increased levels of TNF-α, IL-6, resistin, PAI-1 and leptin, and reduced levels 

of adiponectin affects glycemic homeostasis, vascular endothelial function, and 

the coagulation system, thus accelerating atherosclerosis. Adipokines and a lowgrade 

inflammatory state may be the link between the metabolic syndrome with 

its cluster of obesity and insulin resistance and cardiovascular disease. 

In fact, atherosclerosis is now recognized as an inflammatory process of the 

arterial wall. Monocytes adhere to the endothelium and then migrate into the 

subendothelial space where they become foam cells loaded with oxidized lipoproteins. 

Foam cell production of metalloproteinases leads to rupture of the 

atherosclerotic plaque’s fibrous cap and then to rupture of the plaque itself. 32 

Thus, an inflammatory process accounts for both the development and evolution 

of atherosclerosis. 

In this inflammatory process, adipokines play multiple roles. TNF-α activates 

the transcription factor nuclear factor-κβ, with subsequent inflammatory changes 

in vascular tissue. These include increased expression of intracellular adhesion 

molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, 33,34 which 

enhances monocyte adhesion to the vessel wall, greater production of MCP-1 

and M-CSF from endothelial cells and vascular smooth muscle cells, 35,36 and upregulated 

macrophage expression of inducible nitric oxide (NO) synthase, interleukins, 

superoxide dismutase, etc. 37,38 Leptin, especially in the presence of high 

glucose, stimulates macrophages to accumulate cholesterol. 39 IL-6 exerts proinflammatory 

activity in itself and by increasing IL-1 and TNF-α. 40 Importantly, 

IL-6 also stimulates liver production of C-reactive protein which is considered a 

predictor of atherosclerosis. 41 IL-6 may also influence glucose tolerance by regulation 

of visfatin. Visfatin, a newly discovered adipocytokine in human visceral 

fat, exerts insulin-mimetic effects in cultured cells and lowers plasma glucose 

levels in mice through activation of the insulin receptor. 42 

PAI-1 concentrations regulated by transcription factor nuclear factor-κβ are 

abnormally high in hyperglycemia, obesity, and hypertriglyceridemia, 43 because 

of increased PAI-1 gene expression. 44 PAI-1 inhibits fibrin clot breakdown, 

thereby favoring thrombus formation upon ruptured atherosclerotic plaques. 45 In 

humans, circulating PAI-1 levels correlate with atherosclerotic events and mortality, 

and some studies suggest PAI-1 is an independent risk factor for coronary 

artery disease. 46 Angiotensinogen is a precursor of angiotensin II (AngII), which 

stimulates ICAM-1, VCAM-1, MCP-1, and M-CSF expression in vessel wall 

cells. 47 AngII also reduces NO bioavailability 48 with loss of vasodilator capacity 

and increased platelet adhesion to vessel walls. 

In humans, endothelial dysfunction is indicative of the preclinical stages of 

atherosclerosis and is prognostic of future cardiovascular events. 49,50 High concentrations 

of pro-inflammatory adipokines may contribute to development of 

endothelial dysfunction. At this stage of disease, the role of resistin is particularly 

interesting. In vitro studies show resistin activates endothelial cells which, when


incubated with recombinant human resistin, release more endothelin-1 and 

VCAM-1. 51 Recombinant human resistin is also reported to induce higher expression 

of mRNA of VCAM, ICAM-1, and pentraxin-3 from endothelial cells, 52 thus 

expressing a biochemical pattern of dysfunctional endothelium. Finally, resistin 

also induces proliferation of aortic smooth muscle cells. 53 In asymptomatic 

patients with family histories of coronary heart disease, plasma resistin levels are 

predictive of coronary atherosclerosis even after control for other established risk 

factors. 54,55 

CONCLUSION 

The molecular effects of adipokines represent a challenging area of research and 

in-depth understanding of their pathophysiology and molecular actions will 

undoubtedly lead to the discovery of effective therapeutic interventions. Reducing 

adipose tissue mass and consequently adipokine concentrations will prevent the 

metabolic syndrome and, if the hypothesis of adipokine-related linkage with 

atherosclerosis is proven, help prevent the development of atherosclerosis. Despite 

the new findings in the field of adipokines, researchers are still led to focus back 

on obesity as an essential primary target in the continued effort to reduce the risk 

of developing the metabolic syndrome and type 2 diabetes with its associated 

cardiovascular complications. 

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8 

CONTENTS 

Post-Prandial Endothelial 

Dysfunction, Oxidative 

Stress, and Inflammation 

in Type 2 Diabetes 

Antonio Ceriello 

Abstract .............................................................................................................123 

Introduction .......................................................................................................124 

Possible Role of Hyperglycemic Spikes in Cardiovascular Diseases..............124 

Fasting Hyperglycemia and Cardiovascular Disease..............................124 

Post-Prandial Hyperglycemia and Cardiovascular Disease: 

Epidemiological Evidence...........................................................125 

Post-Prandial Hyperglycemia and Cardiovascular Disease: 

Intervention Studies.....................................................................126 

Mechanisms Involved........................................................................................127 

Post-Prandial Hyperglycemia and Oxidative and Nitrosative Stress...............128 

Conclusions .......................................................................................................131 

References .........................................................................................................132 

ABSTRACT 

Increasing evidence suggests that the post-prandial state is a contributing factor to 

the development of atherosclerosis. In diabetes, the post-prandial phase is characterized 

by a rapid and large increase in blood glucose levels. The possibility that 

the post-prandial “hyperglycemic spikes” may be relevant to the onset of cardiovascular 

complications recently received much attention. Epidemiological studies 

and preliminary intervention studies have shown that post-prandial hyperglycemia 

is a direct and independent risk factor for cardiovascular disease. Most of the 

cardiovascular risk factors are modified in the post-prandial phase in diabetic subjects 

and directly affected by an acute increase of glycemia. The mechanisms 

through which acute hyperglycemia exerts its effects may be identified in the 

123


production of free radicals. This alarmingly suggestive body of evidence for a 

harmful effect of post-prandial hyperglycemia on diabetic complications has been 

sufficient to influence guidelines from key professional scientific societies. Correcting 

post-prandial hyperglycemia may form part of a strategy for the prevention and 

management of cardiovascular diseases in diabetes. 

INTRODUCTION 

Diabetes mellitus is characterized by a high incidence of cardiovascular disease 1 

and poor control of hyperglycemia appears to play a significant role in the 

development of cardiovascular disease in diabetes. 2 Increasing evidence indicates 

that the post-prandial state is an important contributing factor to the development 

of atherosclerosis. 3 In diabetes, the post-prandial phase is characterized by a rapid 

and large increase in blood glucose levels. The possibility that these post-prandial 

“hyperglycemic spikes” may be relevant to the pathophysiology of late diabetic 

complications is recently receiving much attention. 

In this chapter, epidemiological data and preliminary results of intervention 

studies indicating that post-prandial hyperglycemia represents an increased risk 

for cardiovascular disease are surveyed. The proposed mechanisms involved in 

this effect are summarized. 

POSSIBLE ROLE OF HYPERGLYCEMIC SPIKES IN 

CARDIOVASCULAR DISEASES 

FASTING HYPERGLYCEMIA AND CARDIOVASCULAR DISEASE 

Over the last 10 years, many studies showed an independent relationship between 

cardiovascular diseases and glycemic control in patients with type 2 diabetes. 2 

These studies involved thousands of subjects, often newly diagnosed, who were 

followed up for periods ranging from 3.5 to 11 years and were evaluated on the 

basis of various cardiovascular end-points. 2 It is necessary to underline that the 

majority of these studies used a single baseline fasting glycemic value or a single 

value of glycated hemoglobin A1c (HbA1c) to predict cardiovascular events 

occurring many years later. 

For instance, the observational version of the United Kingdom Prospective 

Diabetes Study (UKPDS) showed that the mean HbA1c value was a good predictor 

of ischemic heart disease. 4 In particular, multivariate analysis showed that 

for each 1% increment in HbA1c there was an approximate 10% increase in the 

risk of coronary heart disease. 4 This evidence is not substantially different compared 

with the results of the interventional version of the UKPDS. In this trial, 

even though the result was not significant (p

Dysfunction, Stress, and Inflammation in Type 2 Diabetes 125 

improving insulin resistance, may have significantly improved the “cluster” of 

cardiovascular risk factors associated with insulin resistance. 

The relationship existing between macroangiopathy and fasting plasma glucose 

or HbA1c is weaker than that observed with microangiopathy. 2 This was 

found in cross-sectional or longitudinal studies. These data support the hypothesis 

that fasting plasma glucose or HbA1c alone is unable to describe thoroughly 

the glycemic disorders occurring in diabetes and its impact on cardiovascular 

disease. In addition to fasting glycemia and HbA1c, emphasis has recently been 

given to the relationship between post-prandial hyperglycemia and cardiovascular 

diseases. 

POST-PRANDIAL HYPERGLYCEMIA AND CARDIOVASCULAR DISEASE: 

EPIDEMIOLOGICAL EVIDENCE 

The oral glucose tolerance test (OGTT) has been used mostly in epidemiological 

studies that attempt to evaluate the risk of cardiovascular disease. The main 

advantage of the OGTT is its simplicity: a single plasma glucose measurement 

2 hours after a glucose load determines whether glucose tolerance is normal, 

impaired, or indicates overt diabetes. 

The caveats of the OGTT are numerous because 75 or 100 g glucose is almost 

never ingested during a meal and, more importantly, many events associated with 

ingesting a pure glucose solution do not incorporate the numerous metabolic 

events associated with eating a mixed meal. Moreover, the relationship between 

glycemia and meal content is contingent upon the contents of the meal. 7 However, 

it has recently been demonstrated that the level of glycemia reached 2 hours after 

an OGTT was closely related to the level of glycemia after a standardized meal 

(mixed meal in the form of wafers containing oat fractionation products, soy 

protein, and canola oil sweetened with honey: 345 kcal, 10.7 g fat, 12.1 g protein, 

8.9 g simple sugars, 41.1 g starch, and 3.8 g dietary fiber), suggesting that the 

OGTT may represent a valid tool to reveal altered carbohydrate metabolism 

during a meal. 8 Interestingly, the correlation is more consistent for the values of 

glycemia in the impaired glucose tolerance range. 8 

From an epidemiological point of view, the Hoorn Study, 9 the Honolulu Heart 

Study, 10 the Chicago Heart Study, 11 and the more recent DECODE Study 12 have 

clearly shown that glucose serum levels 2 hours after oral challenge with glucose 

are powerful predictors of cardiovascular risk. This evidence is confirmed also 

by two important meta-analyses: the first by Coutinho et al. examined studies of 

95,783 subjects. 13 The second, covering more than 20,000 subjects, pooled the 

data of the Whitehall Study, the Paris Prospective Study, and the Helsinki Policemen 

Study. 14 

The possible role of post-prandial hyperglycemia as an independent risk factor 

has also been supported by the Diabetes Intervention Study showing how in type 

2 diabetics post-prandial hyperglycemia predicts infarction 15 and by another 

study associating post-prandial hyperglycemia levels with medio-intimal carotid 

thickening. 16


TABLE 8.1 

Epidemiological Studies Showing Association of Post-Prandial 

Hyperglycemia with Risk of CVD and Mortality 

Study Result 

Intriguing evidence comes from a study that demonstrates how medio-intimal 

carotid thickening is correlated not only with post-prandial glucose serum level 

but particularly with glycemic spikes during the OGTT. 17 A post-challenge glucose 

spike was defined as the difference between the maximal post-challenge 

glucose level during OGTT, irrespective of the time after glucose challenge, and 

the level of fasting plasma glucose. 17 Epidemiological studies are summarized in 

Table 8.1. 

Indirect evidence of the unfavorable role of acute hyperglycemia on cardiovascular 

diseases is also available. Hyperglycemia during a cardiovascular acute 

event is unfavorable from a prognostic view in cases both of myocardial 

infarction 18,19 and stroke. 20,21 A worst prognosis has been demonstrated for both 

cases in diabetic and non-diabetic subjects. 18–21 

Regarding infarction, it has been recently demonstrated by a meta-analysis 

that there is a continuous correlation between glucose serum levels and the 

seriousness of prognosis even in non-diabetic subjects, 22 while intensive insulin 

treatment during acute myocardial infarction reduced long-term mortality in 

diabetic patients. 23 This is consistent with the evidence that an acute increase of 

glycemia significantly prolongs the QT in normal subjects 24 and that during 

myocardial infarction increased glucose level is capable of inducing such electrophysiological 

alterations as to favor the occurrence of arrhythmias whose 

outcomes could even be fatal. 25 

POST-PRANDIAL HYPERGLYCEMIA AND CARDIOVASCULAR DISEASE: 

INTERVENTION STUDIES 

Ref. 

No. 

Hoorn Study 2-hour glucose better predictor of mortality than HbA1c 9 

Honolulu Heart Program 1-hour glucose predicts coronary heart disease 10 

Chicago Heart Study 2-hour post-challenge glucose predicts all cause mortality 11 

DECODE High 2-hour post-load blood glucose associated with 

increased risk of death independent of fasting glucose 

12 

Coutinho M et al. 2-hour glucose associated with CHD 13 

Whitehall, Paris, and 2-hour post-challenge glucose predicts all cause and CHD 14 

Helsinki Studies 

mortality 

Diabetes Intervention Study Post-meal, but not fasting glucose, associated with CHD 15 

One of the major concerns about the role of post-prandial hyperglycemia in 

cardiovascular disease has been, until now, the absence of intervention studies. 

That situation is changing.


The STOP-NIDDM trial has presented data indicating that treatment of 

patients who have IGT with acarbose, an α-glucosidase inhibitor that specifically 

reduces post-prandial hyperglycemia, is associated not only with a 36% reduction 

in the risk of progression to diabetes 26 but also with a 34% risk reduction in the 

development of new cases of hypertension and a 49% risk reduction in cardiovascular 

events. 27 In addition, in a subgroup of patients, carotid intima media 

thickness (CIMT) was measured before randomization and at the end of the 

study. 28 Acarbose treatment was associated with a significant decrease in the 

progression of intima media thickness, an accepted surrogate for atherosclerosis. 28 

In a recent meta-analysis of type 2 diabetic patients, acarbose treatment was 

associated with significant reductions in cardiovascular events, even after adjusting 

for other risk factors. 29 Finally, very recently, the effects of two insulin 

secretagogues, repaglinide and glyburide, known to have different efficacies for 

post-prandial hyperglycemia, CIMT, and markers of systemic vascular inflammation 

in type 2 diabetic patients, have been evaluated. 30 After 12 months, 

post-prandial glucose peak measurements were 148 ± 28 mg/dL in the repaglinide 

group and 180 ± 32 mg/dL in the glyburide group (p 0.020 mm, was observed in 52% of diabetics receiving repaglinide and in 

18% of those receiving glyburide (p


2 diabetic patients and that the decrease correlated inversely with the magnitude 

of post-prandial hyperglycemia. 39 

The possible role of hyperglycemia in the activation of blood coagulation has 

previously been reviewed. 40 It emerges that acute glycemic variations are matched 

with a series of alterations of coagulation that are likely to cause a thrombosis. 

This tendency is documented by studies that demonstrate how inducing hyperglycemia 

produces a shortening of fibrinogen half-life 41 and increases in fibrinopeptide 

A, 42,43 fragments of prothrombin, 44 factor VII, 45 and platelet aggregation 46 

in both normal and diabetic subjects. These data indicate that coagulation is 

activated during experimental hyperglycemia. It is interesting that the over-production 

of thrombin by post-prandial hyperglycemia in diabetics has already been 

already documented. The phenomenon is strictly dependent on the glycemic levels 

reached. 47 

Adhesion molecules regulate the interactions of endothelium and leukocytes. 

48 They participate in the process of atherogenesis because their greater 

expression would imply an increase in the adhesion of leukocytes (monocytes in 

particular) to the endothelium. 49 It is well known that this is considered one of 

the early stages of the process leading to atheromatous lesions. Among the various 

pro-adhesive molecules, ICAM-1 has received particular interest. Increases in the 

circulating form of this molecule have been demonstrated in subjects with vascular 

disease 50 and with diabetes mellitus with or without vascular disease. 51,52 

These increases have been considered indications of the activation of the atherogenic 

process. 

The soluble form of ICAM-1 is stored in cells and can be quickly expressed 

outside them as a consequence of various stimuli. It has been demonstrated that 

acute hyperglycemia in both normal and diabetic subjects is a sufficient stimulus 

for the circulating level of ICAM-1 to increase, thus activating one of the first 

stages of the atherogenic process. 53,54 

The concept of atherosclerosis as an inflammatory disease even in diabetes 

is now well established. 55 Studies support the evidence that acute hyperglycemia 

during a hyperglycemic clamp 56 or in a post-prandial state 57 can increase the 

production of plasma IL-6, TNF-α, and IL-18. 

POST-PRANDIAL HYPERGLYCEMIA AND OXIDATIVE AND 

NITROSATIVE STRESS 

Recent studies demonstrated that hyperglycemia induces an over-production of 

superoxide by the mitochondrial electron-transport chain. 58 Superoxide overproduction 

is accompanied by increased nitric oxide generation due to eNOS and 

iNOS in an uncoupled state — a phenomenon favoring the formation of peroxynitrite, 

a strong oxidant that in turn damages DNA. 59 DNA damage is an obligatory 

stimulus for the activation of the poly(ADP-ribose) polymerase nuclear 

enzyme. Poly(ADP-ribose) polymerase activation in turn depletes the intracellular 

concentration of its substrate NAD + , slowing the rate of glycolysis, electron


Hyperglycemia 

Mitochondria 

− O2 PKC NF-kB 

NAD(P)H 

oxidase 

− 

O2 PARP 

NAD + 

DNA damage 

iNOS eNOS 

NO 

GAPDH 

Peroxynitrite 

Nitrotyrosine 

Endothelial dysfunction 

Polyol pathway 

AGE formation 

Hexosamine Flux 

Adhesion molecules 

Proinflammatory 

Cytokines 

Diabetic 

complications 

FIGURE 8.1 In endothelial cells, glucose can pass freely in an insulin-independent manner 

through the cell membrane. Intracellular hyperglycemia induces over-production of 

superoxide at the mitochondrial level. Over-production of superoxide is the first and key 

event in the activation of all other pathways involved in the pathogenesis of diabetic 

complications such as polyol pathway flux, increased AGE formation, activation of protein 

kinase C and NF-kB, and increased hexosamine pathway flux. O 2 – reacting with NO 

produces peroxynitrite (ONOO – ). Superoxide over-production reduces eNOS activity but 

through NF-kB and PKC activates NAD(P)H and increases iNOS expression: the final 

effect is an increased nitric oxide generation. This condition favors the formation of the 

strong peroxynitrite oxidant that in turn produces in iNOS and eNOS an uncoupled state, 

resulting in the production of superoxide rather than NO, and damages DNA. DNA damage 

is an obligatory stimulus for the activation of the poly(ADP-ribose) polymerase nuclear 

enzyme. Poly(ADP-ribose) polymerase activation in turn depletes the intracellular concentration 

of its substrate NAD + , slowing the rate of glycolysis, electron transport, and 

ATP formation, and also produces an ADP ribosylation of the GAPDH. This process 

results in acute endothelial dysfunction in diabetic blood vessels that contributes to the 

development of diabetic complications. NF-kB activation also induces a pro-inflammatory 

condition and adhesion molecule over-expression. All these alterations produce the final 

picture of diabetic complications. 

transport, and ATP formation and also produces an ADP ribosylation of the 

GAPDH. 59 These processes result in acute endothelial dysfunction in diabetic 

blood vessels that, convincingly, contributes to the development of CVD. 59 These 

pathways are summarized in Figure 8.1. 

Several indirect and direct approaches support the concept that acute hyperglycemia 

works through the production of oxidative and nitrosative stress. Indirect 

evidence is obtained through the use of antioxidants. The fact that antioxidants


can hinder some of the effects acutely induced by hyperglycemia–endothelial 

dysfunction, 36,60,61 activation of coagulation, 44 plasmatic increase of ICAM-1, 53 

and interleukins 57 suggests that the action of acute hyperglycemia is mediated by 

the production of free radicals. 

Direct evidence is linked to estimates of the effects of acute hyperglycemia 

on oxidative stress markers. It has been reported that during oral glucose challenge, 

a reduction of the antioxidant defenses was observed. 62–64 This effect can 

be observed in other physiologic situations related to eating a meal. 65 The role 

of hyperglycemia is highlighted by the results of a study involving two different 

meals resulting in two different levels of post-prandial hyperglycemia. The greater 

drop in antioxidant activity was linked with the higher levels of hyperglycemia. 34 

The evidence that LDLs are more prone to oxidation in the post-prandial phase 

in diabetics matches these results. 33 Even in this situation, higher levels of hyperglycemia 

are matched with a greater oxidation of LDLs. 34 Finally, the evidence 

that managing post-prandial hyperglycemia can reduce post-prandial generation 

of endothelial dysfunction 66 and oxidative and nitrosative stress 67 strongly supports 

this hypothesis. 

Interesting and new data are available on the possible generation of nitrosative 

stress during post-prandial hyperglycemia. The simultaneous over-generation of 

NO and superoxide favors the production of a toxic reaction product, the peroxynitrite 

anion. 68 The peroxynitrite anion is cytotoxic because it oxidizes sulfhydryl 

groups in proteins, initiates lipid peroxidation, and nitrates amino acids such as 

tyrosine which affects many signal transduction pathways. 68 The production of 

peroxynitrite can be indirectly inferred by the presence of nitrotyrosine, 68 and it 

has recently been reported that nitrotyrosine is an independent predictor of CVD. 69 

Several pieces of evidence support a direct role of hyperglycemia in favoring 

nitrotyrosine over-generation. Nitrotyrosine formation was detected in the artery 

walls of monkeys during hyperglycemia 70 and also in the plasma of healthy 

subjects during hyperglycemic clamp 71 or OGTT. 72,73 Hyperglycemia was also 

accompanied by nitrotyrosine deposition in perfused working hearts from rats, 

and was reasonably related to unbalanced production of NO and superoxide 

through iNOS over-expression. 74 Nitrotyrosine formation is followed by the 

development of endothelial dysfunction in both healthy subjects 71,72 and in coronaries 

of perfused hearts. 74 This effect is not surprising because it has been 

shown that nitrotyrosine can also be directly harmful to endothelial cells. 75 

Dyslipidemia also is a recognized risk factor for cardiovascular disease in 

diabetes 76 and post-prandial hyperlipidemia contributes to this risk. 77 In non-obese 

type 2 diabetes patients with moderate fasting hypertriglyceridemia, atherogenic 

lipoprotein profiles were amplified in the post-prandial state. 78 Such observations 

have raised the question of whether post-prandial hyperlipidemia, which rises 

concomitantly with post-prandial hyperglycemia, is the true risk factor. 79 Evidence 

suggests that post-prandial hypertriglyceridemia and hyperglycemia independently 

induce endothelial dysfunction through oxidative stress. 80 It is now 

well recognized that endothelial dysfunction is one of the first stages and 

earliest markers in the development of cardiovascular disease. 81 Recent studies


demonstrate both independent and cumulative effects of post-prandial hypertriglyceridemia 

and hyperglycemia on endothelial function, with oxidative stress as 

the common mediator. 72,73 This lends credence to the idea of a direct atherogenic 

role for post-prandial hyperglycemia independent of the role of lipids. 

CONCLUSIONS 

The evidence described proves that hyperglycemia can acutely induce alterations 

of normal human homeostasis. It should be noted that acute increases of glucose 

serum level cause alterations in both healthy (normoglycemic) subjects and also 

in diabetic subjects who have basic hyperglycemia. On the basis of this evidence 

we can hypothesize that the acute effects of glucose serum levels can add to those 

produced by chronic hyperglycemia, thus contributing to a final picture of complicated 

diabetes. The precise relevance of this phenomenon is not exactly comprehensible 

and quantifiable at the moment, but based on the tendency to rapid 

variations of hyperglycemia in the lives of diabetic patients, primarily in the 

post-prandial phase, it is proper to think that it may exert an influence on the 

onset of complications. Epidemiological studies 3 and preliminary intervention 

studies seem to support this hypothesis. 27–30 

DCCT in type 1 diabetes 82 and UKPDS in type 2 diabetes 5 both attest to the 

importance of long-term glycemic control through HbA1c for the prevention of 

complications. However, the authors of the DCCT pointed out further that HbA1c 

only is not a sufficient parameter to explain the onset of such complications and 

suggested that post-prandial hyperglycemic excursions could reasonably favor 

the onset of diabetic complications. 82 

Evidence shows that post-prandial glucose serum level is the major determinant 

of HbA1c level after mean daily blood glucose 83–86 and that reducing 

post-prandial hyperglycemia significantly reduces HbA1c levels in type 2 diabetic 

patients. 87,88 On the basis of this evidence, it seems obvious that if post-prandial 

hyperglycemia is important to determine the level of HbA1c, which is fundamental 

to determining the extent of the risk of diabetic complications, it can be 

supposed that post-prandial glucose serum level will favor it as much. 

Evidence also suggests that post-prandial excursions of blood glucose may 

be involved in the development of diabetic complications, particularly cardiovascular 

complications, but are not the only factors. 89,90 Many questions remain 

unanswered regarding the definition of post-prandial glucose and, perhaps most 

importantly, whether post-prandial hyperglycemia has a unique role in the pathogenesis 

of diabetic vascular complications and should be a specific target of 

therapy. 

However, this alarmingly suggestive body of evidence for a harmful effect 

of post-prandial hyperglycemia on diabetic complications has been sufficient to 

influence guidelines from key professional bodies including the World Health 

Organization, 91 the American Diabetes Association, 92 the American College of 

Endocrinology, 93 the International Diabetes Federation, 94 the Canadian Diabetes


Association, 95 and more recently a large task force of European scientific societies 

focusing on cardiovascular disease. 96 

Therefore the real question, as recently underlined also by the American 

Diabetes Association, 97 seems to be that “because CVD is the major cause of 

morbidity and mortality in patients with diabetes, and in type 2 diabetes in 

particular, understanding the impact on CVD events of treatment directed at 

specifically lowering post-prandial glucose is crucial.” Future studies must be 

designed specifically to evaluate this fundamental issue that may significantly 

change the therapeutic approach to diabetes. 

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9 

CONTENTS 

Obesity and 

Inflammation: 

Implications for 



Introduction .......................................................................................................139 

Obesity and Cardiovascular Risk......................................................................141 

Clinical Markers of Inflammation ....................................................................143 

C-Reactive Protein...................................................................................144 

Leptin .....................................................................................................146 

Adiponectin..............................................................................................147 

Tumor Necrosis Factor-α ........................................................................148 

Hemostatic Parameters ............................................................................150 

Resistin ....................................................................................................151 

Other Inflammatory Markers...................................................................152 

Atherosclerosis and Inflammation ....................................................................152 

Obesity and Inflammation.................................................................................154 

Modification of Inflammation in Obesity.........................................................156 

C-Reactive Protein...................................................................................157 

Tumor Necrosis Factor-α ........................................................................159 

Hemostatic Factors ..................................................................................159 

Summary ...........................................................................................................160 

References .........................................................................................................160 

INTRODUCTION 

Age-adjusted mortality rates for cardiovascular disease have been significantly 

declining in the United States over the past 20 years. The encouraging reduction 

is due to a complex interaction of continued improvements in diagnostic imaging, 

medical therapy, and surgical techniques. The pathogenesis of atherosclerosis is 

139


complex and best regarded as a syndrome currently lacking a unifying hypothesis 

that explains all aspects of the initiation and progression of vascular disease. 

However, the recognition and modification of potential cardiovascular risk factors 

that have been statistically related to coronary disease (in particular factors whose 

modification has been demonstrated to reduce clinical event rates in prospective 

controlled clinical trials) have gained credence as a means to reduce the risk from 

atherosclerosis. 

The improvements in cardiovascular morbidity and mortality rates are threatened 

by changing patterns in obesity and diabetes. The incidence and prevalence 

of obesity are significantly increasing in the United States and represent a major 

health hazard due to the independent risks directly related to increased body mass 

index per se in addition to the associated potential risks for the development of 

diabetes, dyslipidemia, and hypertension. 1 

Individuals may be classified as normal, overweight, obese, or morbidly obese 

on the basis of body mass index (weight in kilograms over body surface area in 

square meters). Normality determined by body mass index is considered below 

25. A subject may be classified as overweight if the body mass index falls between 

25 and 30. Obesity is defined as a body mass index in excess of 30 and morbid 

obesity requires a body mass index above 40. 

Recent epidemiologic data concerning the prevalence of obesity in the United 

States utilized the National Health and Nutrition Education Survey (NHANES). 2 

The prevalence of subjects who qualified as overweight or obese in the period 

between years 2003 and 2004 was compared to an earlier phase of the survey 

that analyzed subjects in 1999 and 2000. The prevalence of obesity increased 

from 27.5 to 31.1% in male subjects between the designated time periods. In 

comparison, the prevalence of obesity in women remained relatively constant at 

approximately 33%. The prevalence of overweight and obese children and adolescents 

was disturbingly high (17.1%). The overall prevalence of obesity in the 

general United States population was determined to be 32.2%. 

The increasing prevalence of obesity and related conditions has led to significant 

health and economic concerns due to the dramatic impacts on both direct 

and indirect health care costs. The total economic costs of obesity in the United 

States are estimated to account for expenditures of approximately $60 billion. 3 

Although the interpretation of the actuarial data became controversial after intense 

scrutiny, the Centers for Disease Control previously estimated that approximately 

400,000 deaths per year can be directly attributed to obesity. Furthermore, the 

health risks associated with increased body weight may overtake the quantitative 

impact of tobacco as a contributor to overall cardiac risk assuming the present 

trends continue. 

Obesity had been previously regarded simply as a passive deposition of excess 

energy stored in adipose tissue. However, an increasing body of clinical and 

experimental evidence has implicated adipose tissue as a highly active endocrine 

organ that is intimately involved in a variety of clinically important metabolic 

pathways that subsequently increase the risk for atherosclerosis. Despite a clear 

univariate statistical correlation between increased body mass index and total or

Obesity and Inflammation: Implications for Atherosclerosis 141 

cardiovascular mortality, the independent cause-and-effect relationship is difficult 

to establish with definite certainty due to multiple potential confounding factors 

including degree of physical activity, regional distribution of fat (truncal versus 

gluteal), socioeconomic status, and associated metabolic disorders such as hypertension, 

hyperlipidemia, and diabetes. Age may also play a role in the impact of 

obesity on mortality; the statistical relationship may attenuate over time and 

become less prominent in older age groups. 4 This review will focus on the role 

of the adipocyte in inflammation and the potential impact on cardiovascular risk. 

OBESITY AND CARDIOVASCULAR RISK 

Obesity has been statistically associated with profound reductions in lifespan in 

both men and women. For example, a 20-year-old Caucasian man with a body 

mass index in excess of 45 is estimated to lose 13 years of life due to medical 

problems directly related to obesity. 5 Cardiovascular disease (including cerebrovascular 

accident and sudden cardiac death) is a major component of the 

increased mortality associated with obesity. 

However, significant clinical and statistical problems are encountered in the 

quantification of the impact of obesity on cardiovascular mortality due to the 

common association of a variety of intimately interrelated and frequently coexistent 

metabolic conditions such as diabetes, dyslipidemia, and hypertension. 

Additionally, individuals who are classified as overweight purely on the basis 

of body mass index may be relatively healthy if free of associated metabolic 

disorders. 

The concept that overweight or obese individuals who are physically fit and 

do not have associated metabolic conditions may not demonstrate increased 

cardiovascular risk has been proposed although considerable controversy exists 

due to methodological problems and the lack of large-scale prospective controlled 

clinical trials. 6 However, it is clear that a lean physically fit individual has the 

lowest overall risk of developing cardiovascular disease. The presence and quantitative 

contributions of lifestyle, diet, exercise patterns, and degrees of physical 

fitness are difficult to determine in epidemiologic studies and may potentially 

confound the interpretation of observational data. 

The duration and intensity of physical activity are also significant determinants 

of body weight, glucose tolerance, and blood pressure but the quantitation 

of physical fitness is frequently problematic in large scale observational studies. 

The relationship of cardiovascular fitness, mortality, and obesity is conflicting. 

Studies have demonstrated that the highest relative risk of all-cause mortality was 

evident in obese individuals who were physically unfit. 7 However, subjects who 

were of normal weight and physically unfit appeared to have higher relative risks 

of mortality when compared to obese individuals with optimal cardio-respiratory 

fitness. The concept of the metabolically healthy obese (MHO) individual has 

been proposed as a means to eliminate confounding findings but is difficult to 

define and has not traditionally been evaluated in epidemiologic studies.


Multiple observational trials performed in men have linked physical fitness 

and health benefits with resultant reductions in risk from cardiovascular disease. 8 

The benefits from cardiovascular fitness have also been demonstrated in women. 

However, conflicting results relative to the contributions of physical fitness and 

obesity may be gender-related. The relationship of physical fitness, obesity, and 

cardiovascular events in roughly 1000 women was determined over a 4-year 

observational period. 9 Women who were demonstrated to have low levels of 

physical activity independent of obesity were significantly more likely to have 

major adverse cardiovascular events during the 48-month follow-up period. The 

results were interpreted to imply that physical fitness and metabolic health may 

be more important than obesity per se for the determination of cardiovascular 

risk in women. The presence of conflicting data has generated a significant debate 

concerning the relative role of “fitness versus fatness” in cardiovascular and total 

mortality and has not been completely resolved. 

Diabetes mellitus has been termed a coronary equivalent due to the high 

risks of developing cardiac events in diabetic subjects. The role of physical fitness 

and alteration of body mass index as a means to reduce the risk of developing 

diabetes has been recently analyzed. 10 The relative contribution of body mass 

index as a means to predict the subsequent risk of development of type II diabetes 

appeared to be stronger than the degree of physical activity. Additionally, 

improved physical activity appeared to have little effect upon the relationship 

between changes in body mass index and development of diabetes. However, 

definite clinical endpoints were not evaluated and the method employed to report 

physical activity may not have been optimal. Thus the relative roles of physical 

activity, metabolic health, and obesity are difficult to separate in observational 

studies and represent significant problems for related risk factors such as dyslipidemia 

and hypertension. 

Despite the methodological difficulties in the analysis of the quantitative 

independent contribution of obesity to global cardiovascular risk, it is clear that 

metabolic factors associated with obesity play a major role in the subsequent 

incidence of cardiac events. The term metabolic syndrome describes a constellation 

of cardiovascular risk factors (including truncal obesity) that appear to share 

common metabolic pathways as a means to identify and target high risk individuals 

for aggressive multifactorial therapeutic interventions. The National Cholesterol 

Education Program has established diagnostic criteria and requires any three 

of the five following risk factors 11 to be present in order to make the diagnosis 

of metabolic syndrome (Figure 9.1): 

1. Abdominal obesity is defined as waist circumference measured at the 

umbilicus and is considered to be positive if in excess of 40" in men 

and 35" in women. 

2. Fasting triglycerides must be in excess of 150 mg per deciliter. 

3. Fasting glucose: blood sugar must be in excess of 110 mg per deciliter.


Fasting 

Glucose 

>110 mg 

HDL 

Men 35 

inches 

Blood 

Pressure 

>130 

>85 

FIGURE 9.1 Clinical diagnosis of the metabolic syndrome. 

Triglyceride 

>150 mg/dl 

4. HDL cholesterol must be less than 40 mg per deciliter in men and less 

than 50 mg per deciliter in women. 

5. Blood pressure must be in excess of 130 mm of mercury systolic and 

85 mm of mercury diastolic. 

The clinical utility of the concept of the metabolic syndrome as a specific disease 

entity has been challenged by the American Diabetes Association and the European 

Association for the Study of Diabetes. 12 However, the use of the syndrome 

term does not necessarily imply a specific disease entity but refers to a grouping 

of clinical symptoms or conditions that appear to coexist in individuals out of 

proportion to the prevalence in the general community. The major cardiovascular 

risk factors share multiple metabolic pathways that have been linked to the process 

of atherosclerosis. The Common Soil Hypothesis of atherosclerosis is based on 

the premise that factors such as obesity, hypertension, diabetes, and dyslipidemia 

share pathways that may play a role both in the preclinical manifestations of 

atherosclerosis and the risk of progression to occlusive vascular disease.(Figure 

9.2). The clinical factors required for the definition of the metabolic syndrome 

are characterized by evidence of inflammation and oxidative stress that is demonstrable 

across the spectrum of atherosclerosis and may represent preclinical targets 

for risk stratification or therapeutic interventions. 13 

CLINICAL MARKERS OF INFLAMMATION 

The advent of clinical markers has established a central role for inflammation as 

a primary pathogenetic mechanism in the initiation and progression of atherosclerosis. 

The concept that atherosclerosis is an inflammatory disease has gained 

credence over the past decade. The recognition that obesity is also associated 

with a significant inflammatory component has been established from a large 

body of clinical evidence that emanated from experimental, epidemiologic, 

genetic, and pathologic studies. 

Additionally, markers of inflammation have been utilized to link obesity with 

other cardiovascular risk factors such as diabetes, hypertension, and dyslipidemia.


Sensitive, reproducible, and specific markers of inflammation are necessary to 

establish accurate risk stratification and potentially to monitor response to therapy. 

While the bulk of clinical and experimental evidence has centered around the 

role of C-reactive protein in the inflammatory state associated with obesity and 

atherosclerosis, a number of other pro- and anti-inflammatory proteins have been 

employed for clinical stratification including leptin, tumor necrosis factor-α, 

adiponectin, interleukins-1, -6, and -10, resistin, fibrinogen, plasminogen activator 

inhibitor-1, and others. 

C-REACTIVE PROTEIN 

Oxidative Stress Inflammation 

Endothelial Dysfunction 


FIGURE 9.2 The common soil hypothesis. 

C-reactive protein is one of the original inflammatory markers that was systematically 

analyzed for a potential role in risk stratification for coronary atherosclerosis. 

An extensive body of epidemiologic and clinical trial evidence has been 

accumulated concerning its role in both vascular disease and obesity. Additionally, 

the utility of C-reactive protein as a marker for cardiovascular risk stratification 

has continued to expand as improved methods of analysis have been developed. 

The original assays for C-reactive protein exhibited wide ranges of normality. 

Considerable intra-individual variability was problematic in analyzing the clinical 

relevance of relatively minor changes occurring over time. 

The subsequent availability of high sensitivity assays has allowed the analysis 

of changes of C-reactive protein within the normal range and significantly 

improved the clinical utility of this marker in cardiac risk stratification. Multiple 

epidemiologic studies in primary prevention have demonstrated that a single 

measurement of C-reactive protein is a strong predictor of future vascular events 

and is independent of the traditional cardiac risk factors. 14 C-reactive protein 

levels are increased in several cardiac risk factors (including truncal obesity) 

required by the National Cholesterol Education Program for the diagnosis of the 

metabolic syndrome and has added prognostic information to traditional risk 

stratification. 15 

C-reactive protein has been demonstrated to be an independent predictor of 

future cardiac risk and adds prognostic information obtained from traditional lipid


screening and the Framingham Risk Score in subjects classified as candidates for 

primary prevention. The clinical utility of quantifying C-reactive protein levels 

in risk stratification in secondary prevention is more controversial because these 

subjects require an aggressive multifaceted therapeutic approach that would 

include interventions that lower inflammatory markers. However, C-reactive protein 

has been demonstrated to be an independent predictor of future cardiovascular 

risk in patients with angiographically documented coronary artery disease and is 

also correlated with the number of angiographically determined complex coronary 

artery lesions in subjects with acute coronary syndromes. 16 The role that Creactive 

protein plays in risk stratification in primary prevention has been clearly 

defined over the past decade. However, increasing evidence indicates that it is 

also a direct participant in the atherosclerotic process. 17 

C-reactive protein is a complex molecule composed of 523 kilodalton subunits 

and has been classified as a member of the pentraxin family. The liver is a major 

site of the synthesis of C-reactive protein which is at least partially under the 

modulation of interleukin-6 produced by adipose cells. 18 C-reactive protein was 

determined to be a major component of the immune response system and is 

directly involved in the primary migration of inflammatory cells into the subendothelial 

space. 19 The recognition that C-reactive protein is an active participant 

in the inflammatory process has raised the possibility of a direct role in the 

pathogenesis of atherosclerosis. 

C-reactive protein has been demonstrated to mediate a variety of pro-inflammatory 

and atherosclerotic effects on human aortic endothelial cells following 

recognition and binding to FC gamma receptors (CD 32 and CD 64). 20 It has also 

been demonstrated to be localized in the vascular intima within regions that 

are prone to the development of atherosclerosis. 21 Additionally, C-reactive 

protein deposition precedes the appearance of monocytes in the earliest stages of 

atherosclerosis. 

The monocytes that subsequently give rise to macrophage scavenger cells 

have been demonstrated to express a specific receptor for C-reactive protein. The 

administration of monoclonal antibodies directed at the receptor eliminates Creactive 

protein-mediated monocyte chemotaxis and supports the major role it 

plays in the recruitment of inflammatory cells in coronary artery disease. The 

localization of C-reactive protein within the subendothelial space is also associated 

with the expression of a variety of cellular adhesion molecules, monocyte 

chemotactic protein (MCP-1), and plasminogen activator inhibitor (PAI-1). Creactive 

protein levels thus appear to be intimately involved with the early phases 

of atherosclerosis and are associated with increased risk across the spectrum of 

vascular disease. 

Additionally, C-reactive protein has been demonstrated to be elevated in 

subjects with increased body mass index and obesity which substantiates the role 

of inflammation in obesity. The epidemiologic association of elevated levels of 

C-reactive protein and cardiovascular risk is robust. 22,23 The presence of elevated 

C-reactive protein in obese and insulin-resistant subjects also appears to predict 

the subsequent risk of development of type 2 diabetes which also implicates


inflammation as a potential mechanism in the pathogenesis of the metabolic 

syndrome. 24 

Hygienic measures such as weight loss in obesity have been demonstrated to 

lower circulating levels of inflammatory markers such as C-reactive protein by 

regulating genes involved with the inflammatory responses in white adipose 

tissue. 25 The elevations of C-reactive protein in obesity link the progressive 

accumulation of adipose tissue with inflammation and may provide correlation 

with cardiovascular risk in obese subjects. 

LEPTIN 

Leptin was one of the original hormones isolated from adipose tissue and the 

discovery subsequently initiated extensive investigations that elucidated its role 

in the complex metabolic pathways characterizing obesity. Leptin is directly 

synthesized within adipocytes and is a central physiologic factor in the regulation 

of both appetite and energy expenditure. It plays a physiologic role in the regulation 

of body mass index by increasing energy expenditure and reducing caloric 

intake to maintain metabolic balance. 

Genetic models such as ob/ob mice that lack the gene encoding for the 

synthesis of leptin demonstrate significant increases in body mass index that are 

also associated with insulin resistance and high subsequent risks for development 

of type II diabetes. 26 

Additionally, the administration of leptin in these models has been demonstrated 

to result in reductions in both caloric intake and body mass index. The 

physiologic action of leptin in the regulation of caloric intake and body mass 

requires the presence of normal hormonal levels, receptor function, and signal 

transduction. Rare genetic conditions associated with morbid obesity have been 

demonstrated to be correlated with either reduced levels of circulating leptin or 

associated receptor abnormalities. 27 Leptin has also been utilized as a therapeutic 

agent and its administration to subjects with genetically mediated deficiency states 

resulted in reductions in body mass index plus improvements in both inflammatory 

and metabolic parameters. 28 

However, significant differences are present in the forms of obesity prevalent 

in the general population compared to subjects with genetically mediated congenital 

leptin deficiency. The increased body mass in obese individuals is associated 

with a significant capacity to synthesize leptin due to the presence of large 

numbers of adipocytes. Leptin resistance is common in subjects with coexistent 

insulin resistance, diabetes, and obesity. The precise mechanism that underlies 

the high circulating levels of leptin and resistance to the normal physiologic 

function of this hormone is complex and multifactorial. The presence of progressively 

increasing levels of leptin appears to result in down-regulation of receptor 

number or activity, with subsequent resistance to the normal physiologic activity 

of the hormone. 

The chronic administration of a diet enriched in calories accounted for by 

saturated fat had been demonstrated to be associated with a subsequent increase


in circulating leptin levels despite a progressive expansion of body mass. The 

induced increase in body mass index secondary to caloric excess has been correlated 

with a suppression of signaling for leptin due to a progressively increased 

expression of SOCS-3 (suppressor of cytokine signaling-3) that significantly 

alters the normal physiologic and regulatory effects within hypothalamic centers 

in the brain. 29 

Leptin also has been demonstrated to have significant metabolic effects in 

peripheral tissues that were initially identified in genetic conditions such as 

congenital lipodystrophy. Experimental models with this uncommon genetic 

condition are associated with minimal adipose tissue deposits in peripheral tissues, 

resistance to insulin, and progressive and persistent hyperinsulinemia. The 

administration of leptin in subjects with congenital lipodystrophy was demonstrated 

to restore the metabolic abnormalities to normal. 30 Leptin is now established 

as a major hormone produced by adipocytes and plays a central role in 

the regulation of energy homeostasis, insulin sensitivity, and body mass index. 

ADIPONECTIN 

Adiponectin is an adipocyte-derived protein shown to express beneficial physiologic 

effects in clinical conditions associated with the development of coronary 

artery disease including significant anti-inflammatory effects 31 (Figure 9.3). It has 

been demonstrated to be interrelated with obesity, insulin resistance, endothelial 

function and atherosclerosis. Adiponectin may reverse a variety of inflammatory 

and metabolic abnormalities which are associated with obesity by increasing the 

sensitivity of insulin in the peripheral tissues with resultant improvement in 

glucose homeostasis. 32 

Adiponectin has also been demonstrated to mediate an improvement in endothelial 

function that may be at least partially due to its potent inherent antiinflammatory 

effect. Adiponectin has been shown to reduce the synthesis of the 

potent tumor necrosis factor-α pro-inflammatory cytokine associated with 

endothelial dysfunction. Management of endothelial function associated with 

Antiinflammatory 

Increased 

Angiogenesis 

Adiponectin 

FIGURE 9.3 Adiponectin and atherosclerosis. 

Improved 

Insulin 

Sensitivity 

Improved Endothelial 

Function


dyslipidemia or insulin resistance may be enhanced by therapy with drugs such 

as PPAR ( peroxisome proliferator-activated receptor) agonists such as fenofibrate 

or insulin sensitizers such as thiazolidinediones that also increase adiponectin 

levels. 33,34 Circulating adiponectin levels have been evaluated in obese male and 

female subjects and proved to be inversely related to body mass index although 

the relationship may be more pronounced in women. 35 

Reduction in the circulating level of adiponectin is demonstrable in multiple 

stages of atherosclerosis ranging from endothelial dysfunction to advanced 

obstructive vascular disease. The earliest stage of atherosclerosis is characterized 

by inflammatory cell binding to the dysfunctional endothelium which is mediated 

by the production of cellular adhesion molecules. The production of adhesion 

molecules is mediated by a variety of pro-inflammatory cytokines such as tumor 

necrosis factor-α and has been demonstrated to be reduced by adiponectin. The 

inverse relationship between adiponectin and tumor necrosis factor-α links the 

protective effect of this adipocyte derived protein to the preclinical phases of 

coronary artery disease. 36 

Adiponectin may also play a protective role in more advanced phases of 

vascular obstruction by increasing angiogenesis in chronic ischemia. Experimental 

studies have been performed in an adiponectin knock-out mouse model that 

employed a chronic hind limb ischemia preparation. The depletion of adiponectin 

resulted in a significant impairment of vascular repair of the involved ischemic 

area. Adenovirus-mediated supplementation of adiponectin resulted in a significant 

improvement in angiogenic repair which implicates a potential role for this 

adipocyte-derived protein in the development of collateral flow in chronic 

ischemia. 37 Adiponectin thus has significant anti-inflammatory activity and is 

reduced in obesity and obstructive vascular disease. 

TUMOR NECROSIS FACTOR-α 

Tumor necrosis factor-α is a pro-inflammatory cytokine whose levels are 

increased in such seemingly disparate clinical conditions as collagen vascular 

diseases and congestive heart failure. Modification of the activity of tumor necrosis 

factor by receptor blockers has been utilized as a therapeutic target in rheumatoid 

arthritis. Tumor necrosis factor-α is directly synthesized in adipocytes 

and is felt to play a significant role in the maintenance of both the mass and 

metabolism of adipose tissue. 38 

The localization of the synthesis of tumor necrosis factor-α within the adipose 

tissue also allows for a potential autocrine or paracrine effect that may alter the 

numbers and sizes of adipocytes in addition to systemic pro-inflammatory effects. 

Tumor necrosis factor-α was found to be associated with enhanced apoptosis of 

stem cells that have the potential to develop into mature adipocytes in addition 

to fully differentiated adipocytes. 39 

The mechanism by which tumor necrosis factor-α induces programmed cell 

death is complex and multifactorial. The pro-inflammatory activity of tumor necrosis 

factor-α is associated with a variety of proteolytic enzymes that may be involved


in progressive and irreversible cellular degradation. 40 Additionally, up-regulation of 

the capase gene may also be related to increased levels of tumor necrosis factor-α 

and has been demonstrated to be inhibited by the administration of potent antiinflammatory 

agents such as glucocorticoids (e.g., dexamethasone). 41 

Tumor necrosis factor-α also plays a significant role in lipid metabolism and 

the resultant metabolic fate of circulating lipoproteins. The degradation of triglyceride-rich 

lipoproteins such as very low density lipoprotein is modulated by 

activation of lipoprotein lipase and results in the generation of free fatty acids. 

The local availability of free fatty acids within adipose tissue is a significant 

determinant of the triglyceride contents of individual adipocytes. Free fatty acids 

may be accumulated within adipocytes and subsequently synthesized into 

triglycerides under the enzymatic regulation of Acyl-CoA synthetase. Tumor 

necrosis factor-α may exert significant effects on fatty acid metabolism and lipid 

contents of adipocytes by direct effects on the activity of lipoprotein lipase which 

alters substrate availability for triglyceride synthesis. 

The intrinsic enzymatic activity of lipoprotein lipase is significantly reduced 

by tumor necrosis factor-α with a resultant decrease in the metabolism of triglyceride-rich 

lipoproteins. The impaired degradation of triglyceride-rich lipoproteins 

decreases the potential for enhanced flux of free fatty acids into adipocytes 

and results in the alteration of intracellular lipid concentration. 43 

Additionally, tumor necrosis factor-α is associated with direct down-regulation 

of acyl Co-A synthetase which may also contribute to the degree of triglyceride 

synthesis and lipid accumulation within adipocytes. 43 

The role of tumor necrosis factor-α in the manifestations of inflammation in 

obesity in human subjects is complex. The local synthesis of tumor necrosis 

factor-α in the adipocytes of obese subjects is increased and sustained weight 

loss has been demonstrated to reduce circulating levels. 44 However, conflicting 

results have been reported concerning the relationship of circulating tumor necrosis 

factor-α and body mass index that may be secondary to methodologic problems. 

Additionally, genetic models for obesity have demonstrated that increased 

circulating levels of tumor necrosis factor-α are associated only with extreme 

increases in body weight. 45 

Obesity and increased body mass index are frequently associated with the 

development of insulin resistance and adult onset diabetes. Inflammation has been 

postulated to play a significant role in the pathogenesis of insulin resistance in 

obese subjects. The role of tumor necrosis factor-α in the pathogenesis of insulin 

resistance has been evaluated in experimental studies. Obese subjects with associated 

insulin resistance showed increased circulating levels of tumor necrosis 

factor-α that were compatible with the possibility of a cause-and-effect relationship. 

Tumor necrosis factor-α may induce insulin resistance by several mechanisms 

including down-regulation of the insulin receptor, decreased GLUT 4 

synthesis, and altered lipolytic activity. 46 The interrelationship between obesity 

and tumor necrosis factor is complex and a hypothesis that explains all aspects 

remains elusive due to multiple genetic, gender, and metabolic issues. However,


it is clear that tumor necrosis factor-α plays a central role in adipocyte function 

and mass and may have implications for insulin resistance and diabetes. 

HEMOSTATIC PARAMETERS 

Obesity has been correlated with a variety of abnormalities of the hemostatic 

system including alterations in the levels of fibrinogen, von Willebrand factor, 

and plasminogen activator inhibitor. Fibrinogen, which is primarily synthesized 

in the liver, is intimately related to the coagulation cascade and additionally is 

an acute phase reactant. Fibrinogen is thus closely linked to inflammation and is 

demonstrated to be increased following a variety of inflammatory stimuli. 

Obesity has been demonstrated to be associated with increased circulating 

levels of fibrinogen. 47 However, the relationship between fibrinogen levels and 

body mass index or localization (truncal versus peripheral) of adipose tissue is 

controversial. The metabolic syndrome (for which truncal obesity is one of the 

diagnostic criteria) has been correlated with abnormalities in multiple coagulation 

parameters. Fibrinogen levels are increased in individuals fulfilling criteria for a 

diagnosis of metabolic syndrome which may contribute to the increased risk of 

hypercoagulability associated with the classical risk factors such as hypertension, 

diabetes, and dyslipidemia. 48 

Endothelial dysfunction is associated with a variety of abnormalities in the 

coagulation and fibrinolytic systems. The von Willebrand factor is a large glycoprotein 

that is synthesized at least partially in endothelial cells and may represent 

a clinical marker for endothelial dysfunction. However, the relationship between 

von Willebrand factor and obesity is complex and difficult to elucidate because 

the glycoprotein is not directly synthesized by adipocytes. However, the levels 

of von Willebrand factor antigen have been significantly correlated with the degree 

of visceral fat in subjects who are either overweight or definitely obese. 49 

Obesity is frequently associated with alterations of the balance between 

thrombotic and fibrinolytic parameters. The primary inhibitor of tissue plasminogen 

activation is mediated by plasminogen activator inhibitor-1 (PAI-1) which 

decreases the rate of breakdown of fibrin clots (Figure 9.4). Risk factors such as 

obesity, hypertension, hypertriglyceridemia, and diabetes are frequently characterized 

by elevations of circulating levels of plasminogen activator inhibitor. The 

presence of a relative excess of circulating plasminogen activator inhibitor is 

associated with an increased risk of occlusive intravascular thrombus due to 

impaired fibrinolysis. Plasminogen activator inhibitor is synthesized in response 

to inflammation by endothelial cells, adipocytes, hepatocytes, and platelets. 50 

Additionally, the levels of plasminogen activator inhibitor have been correlated 

with both obesity and increased body mass index which establishes an association 

of obesity, hypercoagulability, and inflammation. 51 

Abnormalities of plasminogen activator inhibitor levels are further correlated 

with the degree of visceral fat, which emphasizes the role of the distribution of 

adipose tissue in cardiovascular risk. 52 The relationship between visceral fat 

accumulation and abnormalities in the fibrinolytic pathway is emphasized by the


fact that reductions in plasminogen activator inhibitor are best correlated with a 

decrease in the amount of visceral adipose rather than body weight per se. 53 

The current definitions of the metabolic syndrome do not include components 

of the hemostatic or fibrinolytic system. However, the risk factors (e.g., diabetes, 

hypertension, obesity, and dyslipidemia) included in the criteria for diagnosis 

have inflammatory bases and are also frequently associated with either hypercoagulability 

or impaired fibrinolysis. 54 Markers of inflammation or fibrinolysis may 

be included in the criteria for metabolic syndrome as further studies evolve and 

more sensitive and specific indicators of cardiac risk are evaluated. 

RESISTIN 

Plasminogen 

Plasminogen 

T-PA 

T-PA 

PAI-I 

Plasmin 

TG 

All 

Lp(a) 

TNF 

Insulin 

Plasmin 

FIGURE 9.4 Endothelial dysfunction and fibrinolysis. 

Fibrinolysis 

Thrombosis 

Resistin is a recently described pro-inflammatory cytokine that is produced within 

adipose tissue and has been linked to both obesity and the risk for development 

of diabetes mellitus. 55 However, the role of resistin in the characteristic obesity 

of humans is controversial despite the compelling data from animal models. 

Resistin is a cysteine-rich polypeptide that is directly synthesized by adipocytes. 56 

Circulating levels of resistin were shown to be increased in experimental 

models that demonstrated impaired insulin sensitivity and diabetes. Additionally, 

circulating resistin levels were reduced by insulin sensitizing agents such as 

thiazolidinediones. 57 The increased levels of resistin in obesity have been demonstrated 

to be generated by direct synthesis from activated macrophages that are 

localized within adipose tissue and regulated by PPAR gamma activators. 58 

The glucose intolerance associated with obesity may also be linked to 

increased levels of resistin via complex metabolic and inflammatory pathways. 

Experimental studies utilizing transgenic mice with high circulating concentrations 

of resistin in the presence of normal weight showed increased glucose 

production in the setting of a hyperinsulinemic–euglycemic clamp. 59 The fact that 

elevated levels of resistin are associated with impaired glucose homeostasis is at 

least partially explained by the associated increased expression of the phosphoenolpyruvate 

carboxykinase hepatic enzyme that serves as a key enzyme in


glucose metabolism. The relationship between resistin and inflammation has been 

demonstrated by an association with inflammatory mediators such as tumor 

necrosis factor-α and C-reactive protein. 60,61 Resistin has been hypothesized to 

play multiple roles in obesity and diabetes secondary to the induction of inflammation 

and a secondary alteration of the metabolism of glucose due to the 

associated insulin resistance. 

OTHER INFLAMMATORY MARKERS 

A variety of other markers have been proposed as clinically relevant parameters 

that have utility in the identification of inflammation and potential cardiovascular 

risk stratification. Interleukin-6, interleukin-10, interleukin-1, adhesion molecules, 

and visfatin have all been identified as potentially valuable clinical markers. 

However, the bulk of clinical and experimental data related to obesity, diabetes, 

and inflammation were derived from the identification of C-reactive protein and 

improved high sensitivity measurements that have allowed accumulation of a 

large body of clinical and experimental evidence that substantiates the role of 

inflammation in obesity 

ATHEROSCLEROSIS AND INFLAMMATION 

The concept of atherosclerosis as an inflammatory disorder is supported by an 

ever-increasing body of pathologic, experimental, epidemiologic, and clinical 

evidence. 62 The delineation of the role that inflammation plays as a primary factor 

in the initiation and progression of vascular disease has led to enhanced understanding 

of the basic biologic properties of atherosclerotic plaques, risk stratification, 

and therapeutic strategies. 

The cellular elements involved in inflammation can be demonstrated to be 

present to variable degrees in multiple stages in the atherosclerotic process. 

Additionally, inflammation also can be related to the classic cardiovascular risk 

factors including obesity. The process by which inflammatory cells are localized 

into the subendothelial space and contribute to atherosclerosis is complex and 

requires cellular recognition, binding, migration, and transformation. The normal 

endothelium is not associated with significant adherence of inflammatory cells. 

However, the dysfunctional endothelium expresses a variety of adhesion molecules 

such as vascular cell adhesion molecule-1 (VCAM-1) and members of the 

selectin (E and P) family. 63 

The progressive expression of adhesion molecules on the dysfunctional endothelium 

provides the means for the recognition, binding, tethering, and transmigration 

of circulating inflammatory cells. The accumulation of inflammatory cells 

is an initial phase of atherosclerosis. Additionally, cytokines and chemokines are 

small proteins that are intimately involved in the transmigration and concentration 

of inflammatory cells into vascular regions prone to develop atherosclerosis. 64 

Cytokines are involved in enhanced migration of several inflammatory cell lines 

of which the monocyte is the prototype example.


Monocyte chemoattractant protein (MCP-1) is a primary regulator of the 

accumulation and concentration of monocytes into the subendothelial spaces in 

the initial stages of atherosclerosis. Monocytes play a significant role in the 

cellular inflammatory response and also can transform into scavenger cells that 

are involved in the recognition and binding of oxidized low density lipoprotein 

with subsequent generation of lipid-laden foam cells. The capacity of the monocyte 

to transform into a macrophage scavenger cell line is under the regulation 

of a variety of colony stimulating factors. 65 The monocyte scavenger receptor 

recognizes both native and modified low-density lipoprotein. However, the binding 

and uptake of non-modified low-density lipoprotein is quantitatively minimal. 

In contrast, low-density lipoproteins modified by processes such as oxidation 

or glycation are progressively internalized at a relatively rapid rate by macrophages 

and are associated with progressive lipid accumulation and the subsequent 

development of atherosclerosis. In contrast to the classic LDL receptor, the 

number or activity of the scavenger receptor is not down-regulated by progressive 

accumulation of intracellular lipid. 

The macrophage that originates from the inflammatory cell line also plays a 

significant role in the progression and ultimate fate of the atherosclerotic plaque. 

The monocyte–macrophage cellular elements are gradually depleted due either 

to apoptosis or the cytotoxic effects of a variety of noxious stimuli. The degradation 

of cellular constituents results in the release of lipids and proteinaceous 

debris into the plaque and thus has implications for vulnerability and rupture. 

Macrophages also play a significant role in the local degree of oxidative stress 

demonstrated to be secondary to the capacity to synthesize cytotoxic reactive 

oxygen species. 66 The concentration and rate of production of reactive oxidative 

species are major contributors to the initiation and progression of atherosclerosis 

effects on lipid oxidation and cellular damage. 

The degree of inflammation also plays a significant role in the vulnerability 

of the atherosclerotic plaque and subsequent risk for rupture. High-grade inflammation 

is associated with a progressive reduction in plaque stability and resultant 

increased vulnerability. Macrophages have the capacity to elaborate a variety of 

proteolytic enzymes that may be involved in the net balance between intraplaque 

matrix synthesis and degradation. Collagenase and gelatinase are matrix metalloproteinases 

that demonstrate the capacity to progressively degrade the stabilizing 

matrix protein constituents and result in a progressively increased risk for 

rupture due to reduction in the tensile strength of the plaque. 67 

Increased plaque vulnerability is characterized by an enhanced risk of erosion 

or frank rupture of the fibrous cap. Destruction of the structural integrity of the 

fibrous cap results in exposure of platelets and coagulation factors to the highly 

thrombogenic lipid-rich core localized within the atherosclerotic plaque. Procoagulants 

such as tissue factor are produced within the lipid-laden foam cell 

and are associated with increased risk for vascular occlusion due to enhanced 

synthesis of activated clotting factors VII and X. 68 Macrophages are also involved 

in the synthesis of inhibitors to plasminogen activators (e.g., PAI-I), resulting in 

a decreased capacity to lyse a potentially occlusive intravascular thrombus. 69


Inflammation is thus a major contributor to all phases of atherosclerosis from 

endothelial dysfunction to acute myocardial infarction. The major cardiac risk 

factors such as diabetes, hypertension, and dyslipidemia are all associated with 

a degree of inflammation and oxidative stress. Recent evidence has centered 

around the role of adipocytes in inflammation and a large body of work relates 

obesity to the inflammatory response. 

OBESITY AND INFLAMMATION 

The major cardiac risk factors frequently coexist and share multiple common 

metabolic pathways. Diabetes, hypertension, the use of tobacco products, and 

dyslipidemia have all been demonstrated to be associated with inflammation. 

However, the concept that obesity is characterized by active inflammation is a 

relatively new concept that initially was not widely accepted. The previous perception 

of obesity was that adipose tissue simply represented a passive storage 

depot for excess energy resulting from increased caloric intake relative to energy 

expenditure. 

Adipose tissue was subsequently demonstrated to be a metabolically active 

endocrine organ and the resultant increase in body mass index was implicated in 

the pathogenesis of diabetes, hypertension, and dyslipidemia. Adipose tissue has 

been shown to be intimately involved in a variety of inflammatory processes and 

the mechanisms have been partially clarified. Adipose tissue is the source of a 

number of cytokines that regulate the production of inflammatory mediators 

including C-reactive protein, tumor necrosis factor-α, interleukin-6, etc. 

Insulin resistance is also thought to play a significant role in the pathogenesis 

of several cardiovascular risk factors in obese subjects. Individuals with elevated 

circulating levels of insulin due to impaired peripheral resistance have increased 

free fatty acid release from adipocytes and enhanced delivery to the liver. The 

increased bioavailability of free fatty acids as a synthetic substrate results in 

enhanced hepatic production of very low-density lipoprotein. Additionally, the 

enzymatic activity of lipoprotein lipase is decreased in obese subjects with insulin 

resistance. The reduced catabolic rates in combination with hepatic overproduction 

of triglyceride-rich particles results in an atherogenic dyslipidemic phenotype 

characterized as the lipid triad (hypertriglyceridemia, low HDL, and small dense 

LDL) (Figure 9.5 and 9.6). 

Additionally, hyperinsulinemia is associated with vascular smooth hypertrophy, 

enhanced sympathetic activity, and increased renal sodium reabsorption with 

the resultant development of systemic hypertension. 70 Adipocytes have been 

implicated as the initial sites of inflammation in obesity and predate the involvement 

of other organ systems. C-reactive protein has been demonstrated to modulate 

the movement of inflammatory cells into adipose tissue via the production 

of a variety of chemokines. Monocyte migration and subsequent transformation 

into macrophages within adipose tissue are key factors in the self-perpetuating 

low-grade inflammation associated with obesity.


Relative Insulin Deficiency 

↓ Lipoprotein Lipase ↑ Free Fatty Acids 

↓ Clearing of VLDL 

FIGURE 9.5 Insulin deficiency and dyslipidemia. 

Increased 

Sympathetic 

Tone 

↑ VLDL ↓ HDL 

Small Dense LDL 



Renal Sodium 

Reabsorption 

Hypertension 

FIGURE 9.6 Insulin levels and hypertension. 

↑ Hepatic Synthesis 

of VLDL 

Vascular 

Hypertrophy 

The demonstration that adipocytes and monocytes share pathways in the 

immune response system emphasizes the role that adipose tissue plays in the 

interaction between inflammation and atherosclerosis. Additionally, the cellular 

precursors to mature adipocytes were demonstrated to have the ability, similar to 

the monocyte–macrophage system, to act as phagocytes. The adipocytes localized 

within fat depots may thus participate in a primary manner in the inflammatory 

response, systemic hypertension, and atherosclerosis. 

The inflammatory response within adipose tissue may at least be partially 

expressed under genetic control. Experimental models that utilized animals with 

genetically transmitted obesity allowed the examination of factors that control 

and modulate inflammatory states associated with increases in body mass. Multiple 

genes have been implicated in the inflammatory response associated with 

obesity. Genes that regulate the monocyte–macrophage system have been demonstrated 

to be present in white adipose tissue. The experimental studies in the 

obese mouse models have demonstrated the expression of a large number of 

transcripts that can be correlated with body mass.


Biochemical markers for macrophages were significantly correlated with both 

body mass and the size of the adipocytes. Analysis of the genes that had the 

highest correlations with obesity demonstrated that approximately 30% were 

involved in encoding for proteins that were characteristic of macrophages. 71 

Tumor necrosis factor-α is a pro-inflammatory cytokine demonstrated to be 

produced by macrophages and is localized within adipose tissue. The potential 

thus exists for a self-perpetuating cycle of increased macrophage infiltration 

coupled with enhanced synthesis of cytokines within adipose tissue and a resultant 

persistent inflammatory milieu. 

The establishment of an inflammatory environment within adipose tissue may 

also play a significant role in the pathogenesis of insulin resistance. However, 

significant differences may exist among genetically mediated obesity, insulin 

resistance, and an increased body mass that is purely secondary to excessive 

dietary intake of calories. Chronic inflammation has been linked to the pathogenesis 

of both type 2 diabetes and insulin resistance. 72 Insulin resistance is due to 

a failure of circulating hormonal levels to mediate the normal metabolic handling 

of glucose despite progressively increased concentrations and has been related 

to the presence of inflammation. However, the underlying pathogenesis and interrelationship 

of the various metabolic pathways is not totally clear. 

Inflammation and macrophage-specific genes have been demonstrated to play 

direct roles in both models of genetic obesity and the obesity associated with a 

high fat diet. 73 Macrophages and inflammatory-specific genes are significantly 

up-regulated in the white adipose tissues of both diet-induced obesity and genetic 

mouse models. The up-regulated genes precede a significant increase in the level 

of circulating insulin. Histologic examination demonstrates that macrophage infiltration 

is present in the white adipose tissue and is also associated with evidence 

of cellular destruction of adipocytes. 

Therapy with rosiglitazone, an insulin sensitizing agent, was associated with 

a significant down-regulation of the macrophage-originated genes. Thus, inflammation 

and macrophage infiltration may play significant roles in both diet-induced 

obesity and genetically induced obesity and result in progressive insulin resistance 

due to a chronic inflammatory and cytotoxic response initiated in adipose tissue. 

MODIFICATION OF INFLAMMATION IN OBESITY 

The identification of obesity as a condition associated with a definite inflammatory 

component has significant clinical implications relative to targets of therapy (i.e., 

modification of body mass index in and of itself, improving the function of 

adipose tissue, and normalizing the levels of inflammatory markers). Therapeutic 

interventions that alter the inflammatory response may also be associated with 

restoration of the normal physiologic function of adipose tissue. 

The initial and primary foci of therapy in obesity involve lifestyle modification 

with restriction of calories and saturated fat coupled with increased energy 

expenditure through physical activity. Increased physical activity has been demonstrated 

to decrease body mass index, improve dysfunctional adipose tissue, and


reduce levels of inflammatory markers. However, subjects who are obese but 

metabolically healthy may require alternative approaches to improve long-term 

cardiovascular risk. As risk factor stratification proceeds beyond tabulation of the 

classical risk factors, modification of inflammatory markers may possibly become 

a primary target for therapy with pharmacologic or lifestyle interventions. 

The role of modification of inflammatory markers as targets for therapy has 

been addressed in a variety of clinical trials utilizing lifestyle or pharmacologic 

interventions. 

C-REACTIVE PROTEIN 

C-reactive protein is a major inflammatory marker and a number of lifestyle or 

pharmacologic interventions have been demonstrated to reduce circulating levels 

of this marker. The effect of weight loss on C-reactive protein has been prospectively 

determined in controlled clinical trials utilizing a variety of dietary interventions. 

The role of implementation of a Mediterranean style diet low in calories 

but associated with an increase in monounsaturated fat coupled with an increase 

in physical activity and designed to decrease body weight by 10% has been 

analyzed. 

Body mass index, interleukin-6, and C-reactive protein were all significantly 

reduced by the combination of diet and lifestyle intervention. Additionally, adiponectin 

was increased, thus demonstrating that diet-induced weight loss and 

enhanced physical activity improved the balance of pro- and anti-inflammatory 

mediators. 74 Risk factors that compromise the components of the metabolic syndrome 

have inflammation as a common denominator. 

The impact of the institution of a Mediterranean style diet on endothelial 

function and markers of inflammation in subjects who fulfill the criteria for the 

diagnosis of the metabolic syndrome has been investigated. The dietary intervention 

consisted of an increase in the consumption of vegetables, whole grains, 

fruits, olive oil, and nuts. The Mediterranean diet resulted in significant reductions 

of C-reactive protein and improvements in insulin resistance. Endothelial function 

score as analyzed by blood pressure and platelet aggregation responses to larginine 

was also improved by dietary therapy. The components of the metabolic 

syndrome were normalized to a greater degree in subjects who were randomized 

to receive intensive dietary interventions. 75 

The effect of statin therapy on inflammatory markers had been presumed to 

be secondary to lipid lowering. However, statins have been clearly demonstrated 

to possess a variety of pleiotropic or non-lipid effects. Statins may play a significant 

role in leukocyte trafficking and T cell activation by binding to a novel 

allosteric site within the β-2 integrin leukocyte function associated antigen-1. 76 

Statin therapy has also been shown to reduce C-reactive protein levels and has 

been correlated with clinical outcomes in prospective clinical studies (albeit in 

post-hoc analyses). 

The role of statin therapy in lipid lowering and alteration of inflammatory 

markers was initially analyzed in the Cholesterol and Recurrent Events (CARE)


trial that prospectively compared pravastatin to placebo in subjects with relatively 

normal cholesterol levels who had suffered acute myocardial infarctions. 77 The 

highest cardiovascular risks occurred in subjects who had significant evidence of 

inflammation and were randomized to receive placebo. Pravastatin therapy 

reduced markers of inflammation (C-reactive protein and serum amyloid A) in 

addition to beneficial effects on lipid profiles. 

The utilization of C-reactive protein in predicting response to statin therapy 

in the primary prevention of atherosclerosis was the subject of the Air Force/Texas 

Coronary Atherosclerosis Prevention Study (AFCAPS/TEXCAPS) analyzing the 

effect of lovastatin in 5742 relatively low risk subjects over 5 years. 78 The effect 

of lovastatin was analyzed after stratification of subjects by the ratio of plasma 

cholesterol:HDL and levels of C-reactive protein. Lovastatin reduced coronary 

events irrespective of C-reactive protein levels in subjects with elevated lipid 

ratios. However, it was also effective in subjects whose lipid ratios were below 

the median but associated with elevated C-reactive protein levels, inferring benefits 

of statin therapy in subjects with evidence of inflammation despite the 

presence of normal lipid levels. 

C-reactive protein is associated with an increased density of the AT-1 angiotensin 

receptor, and modulators of the renin angiotensin system may be associated 

with an anti-inflammatory effect. Losartan, a renin angiotensin receptor blocker, 

has been associated with reductions in levels of C-reactive protein although this 

may not be a class effect. 79,80 

Diabetes and insulin resistance have been correlated with a variety of markers 

of inflammation including C-reactive protein that additionally may play a role in 

pathogenesis. 81 The treatment of insulin resistance and diabetes with insulin 

sensitizing agents such as rosiglitazone may demonstrate beneficial affects on 

both circulating glucose levels and markers of inflammation such as C-reactive 

protein. 82 Statins have been demonstrated to reduce the morbidity and mortality 

in diabetes in multiple large scale trials and are also effective even if baseline 

lipids are relatively normal. 83,84 Additionally, HMG CoA reductase inhibitor therapy 

has been demonstrated to reduce the incidence of diabetes in subjects in 

primary prevention, implicating a possible non-lipid effect of statin therapy. 85 The 

multivariate predictors of the development of diabetes in the West of Scotland 

Coronary Prevention Study were glucose, body mass index, and triglyceride 

levels. Interestingly, randomization to pravastatin therapy resulted in a 30% reduction 

in the incidence of diabetes. The precise mechanism involved in the reduction 

of the incidence of diabetes could not be delineated but a potential benefit from 

an anti-inflammatory effect was postulated. 

Additionally, angiotensin-converting enzyme inhibition has demonstrated 

clinical benefits across the spectrum of atherosclerosis in multiple clinical trials. 

Angiotensin II has been shown to demonstrate a significant inflammatory effect 

and the use of ramipril was demonstrated to decrease the incidence of diabetes 

in the Heart Outcomes Prevention Evaluation (HOPE) study. 86 Additionally, in 

an extension of the original HOPE trial that continued 30 months after the


termination of the initial trial, ramipril use was shown to be associated with 

sustained reductions in the incidence of new onset diabetes. 

TUMOR NECROSIS FACTOR-α 

Tumor necrosis factor-α is well established as a pro-inflammatory cytokine 

although the effects of interventions to modify circulating levels in obesity or 

cardiovascular disease lack the broad data base associated with C-reactive protein. 

However, tumor necrosis factor-α is directly synthesized in adipose tissue and 

may be a target for therapy in obesity. 

Clinical studies focused on the role of weight reduction in obese subjects as 

a means to reduce insulin resistance, utilizing hypocaloric diets and increased 

physical activity, have demonstrated decreases in circulating levels of adipokines 

including tumor necrosis factor-α. 87 However, the individual studies were small 

and the reduction in tumor necrosis factor approached but did not reach statistical 

significance. 

Hypolipidemic therapy with statins has been demonstrated to reduce tumor 

necrosis factor-α levels. Interestingly, the reduction in levels also correlated with 

a significant reduction in matrix metalloproteinase levels, which provides a link 

among statin therapy, inflammation, and plaque stability. 88 The increase in tumor 

necrosis factor-α levels associated with obesity and insulin resistance may be 

successfully managed by treatment with PPAR gamma agonists. 

The anti-inflammatory effect of rosiglitazone has been evaluated in both 

diabetic and non-diabetic obese subjects. Tumor necrosis factor-α levels were 

reduced in obese subjects compatible with significant anti-inflammatory effects 

of the PPAR gamma agonists. Tumor necrosis factor has been established as a 

valuable marker of inflammation and may be reduced by a variety of lifestyle 

and pharmacologic interventions that improve lipids, insulin sensitivity, and blood 

pressure in addition to potential anti-inflammatory effects. However, further clinical 

trials are necessary to demonstrate the clinical effects of modifying circulating 

levels of tumor necrosis factor-α. 

HEMOSTATIC FACTORS 

Hemostatic factors such as fibrinogen and plasminogen activator inhibitor are 

increased in obesity and are also linked to inflammation. However, lifestyle and 

pharmacologic interventions produced variable effects on these parameters and 

a consensus view of beneficial interventions has not been established due to 

conflicting results. However, in some studies, statin therapy was demonstrated to 

improve hemostatic parameters. 

Atorvastatin has been demonstrated to improve global fibrinolytic activity 

and reduce plasminogen activator inhibitor coupled with an increase in tissue 

plasminogen activator levels in hypercholesterolemic patients. 89 The effect of 

simvastatin on hemostatic and fibrinolytic factors has been studied in diabetic 

patients. In addition to the beneficial effects on total and low-density lipoprotein


cholesterol levels, simvastatin was demonstrated to reduce circulating plasminogen 

activator inhibitor concentration and prothrombinase activity. 90 

Additionally, lifestyle interventions have been demonstrated to beneficially 

alter fibrinolytic activity in some studies of obese patients. Reduction in weight 

and increased physical activity in obese subjects with insulin resistance have been 

demonstrated to result in improvement in levels of plasminogen activator inhibitor. 

91 Hemostatic factors may be related to an imbalance in fibrinolytic activity 

and impaired capacity to lyse intravascular thrombi. While hemostatic factors 

represent attractive targets in obesity due to their relationship with inflammation 

and may be modified by pharmacologic interventions or lifestyle changes, a 

consensus view regarding their role as a modifiable risk factor remains controversial 

and will require larger prospective studies. 

SUMMARY 

The incidence and prevalence of obesity are markedly increasing in the industrialized 

world and have reached epidemic proportions. Obesity had been regarded 

as a passive depot for excess energy. Recent studies have demonstrated that 

adipose tissue is a dynamic and metabolically active organ system with multiple 

endocrine functions. Obesity is significantly interrelated with a variety of classical 

cardiac risk factors including diabetes, hypertension, dyslipidemia, and hemostatic 

abnormalities. Adipose tissue has been demonstrated to produce a number 

of pro-inflammatory cytokines that establish a self perpetuating low-grade inflammatory 

state. Lifestyle modifications such as caloric restriction and increased 

physical activity have been demonstrated to reduce both body weight and a variety 

of inflammatory markers. 

Pharmacologic interventions such as statin therapy, modulators of the renin 

angiotensin system, and insulin sensitizers have been demonstrated to exhibit not 

only effects on blood pressure, lipids and glucose, but they also significantly 

reduce inflammatory mediators. Thus obesity should be viewed as a highly active 

metabolic state with strong components of inflammation and oxidative stress that 

predispose a patient to coronary artery disease and should represent an active 

target for interventions designed to reduce risks of coronary artery disease. 

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10 Oligomeric 

Composition of 

Adiponectin and 

Obesity 

CONTENTS 

T. Bobbert and Joachim Spranger 

Introduction .......................................................................................................167 

Adipocytes and Adiponectin.............................................................................168 

Biological Activities of Adiponectin Multimers ..............................................169 

Results of Weight Loss .....................................................................................171 

Central Nervous System Effects.......................................................................172 

Summary ...........................................................................................................173 

References .........................................................................................................173 

INTRODUCTION 

Obesity is among the most frequently encountered metabolic diseases worldwide. 

Moreover, its incidence and prevalence are rising rapidly. 1,2 More than half the 

world population is considered overweight. 3 Being overweight constitutes a health 

risk because it is associated with several co-morbidities including dyslipidemia, 

hypertension, type 2 diabetes, and atherosclerotic cardiovascular disease. 4.5 Adipose 

tissue was initially believed to be only a fat storage organ, but it is now 

acknowledged to be an active participant in energy homeostasis and other physiological 

functions. Adipose tissue is known to express and secrete a variety of 

novel adipocytokines that have been implicated in the development of insulin 

resistance and atherosclerosis. 6,7 Dysregulation of adipocytokine production is 

directly involved in the pathophysiology of metabolic syndrome, and normalization 

of plasma concentrations of adipocytokines reverses the phenotype of metabolic 

syndrome. 8,9 

167


ADIPOCYTES AND ADIPONECTIN 

Adipocytes secrete a variety of polypeptides such as leptin, resistin, and adiponectin. 

Adiponectin, the gene product of the adipose tissue’s most abundant 

gene transcript, 10 may be a link between obesity and the development of insulin 

resistance. cDNAs for adiponectin were first identified independently in different 

mouse cells 11,12 and by large-scale random sequencing of a 3′-directed human 

adipose tissue cDNA library. 10 Subsequently, adiponectin was purified from 

human plasma, 13 where it circulates in considerable concentrations. Especially in 

the regulation of the glucose and lipid metabolism, adiponectin has been shown 

to play an important role. 11–13 

Unlike other adipose-derived proteins, plasma levels of adiponectin have been 

found to be decreased in a number of deranged metabolic states including obesity, 

14 dyslipidemia, 15 type 2 diabetes, and insulin resistance. 12,16,17 Many studies 

have demonstrated that reduced circulating adiponectin levels can be partially 

elevated after induction of weight loss in obese and insulin-resistant subjects. 17,18 

Apart from a connection to obesity or diabetes, further parameters like age, sex 

hormones, and glucocorticoids may play parts in the regulation of adiponectin 

levels. 19,20 

Adiponectin is composed of a carboxyl-terminal globular domain and an 

amino-terminal collagenous domain. 21,22 It belongs to the soluble collagen superfamily 

and has structural homology with collagen VIII, X, complement factor 

C1q, 16 and the tumor necrosis (TNF) family. 10,21 This kind of structure is known 

to form characteristic multimers. 23,24 

Gel filtration and velocity gradient sedimentation studies revealed adiponectin 

circulating in serum to form several different molecular weight species; the largest 

species was more than several hundred kilodaltons in size. 11,13,14 Scherer et al. 

noted that adiponectin from 3T3-L1 adipocytes forms trimers, hexamers, and 

larger multimers. 11 Tsao et al. and Arita et al. analyzed multimer formation of 

adiponectin in serum by gel filtration chromatography and showed adiponectin 

to be separable into three species. 14,25 

Kadowaki et al. showed a method of evaluating the multimer formation of 

adiponectin. 26 Using SDS-PAGE under non-reducing and non-heat-denaturing 

conditions, they separated multimers of adiponectin from various sources into 

three species; LMW trimers, MMW multimers, and HMW multimers. This classification 

is not used in all publications and depends partially on the determination 

method used. Also the MMW fractions or hexamers are added to the LMW 

oligomers and predictions or correlations were calculated by the ratio of HMW 

to MMW plus LMW species. 

Oligomer formation of adiponectin depends on the disulfide bond formation 

mediated by Cys-39. 27 Adiponectin was reported to be an α-2,8-linked disialic 

acid-containing glycoprotein, although the biological functions of the disialic acid 

epitope of adiponectin remain to be elucidated. 28 The regulation of adiponectin 

multimerization and secretion occurs also via changes in post-translational modifications 

(PTMs). PTMs identified in murine and bovine adiponectin include

Oligomeric Composition of Adiponectin and Obesity 169 

hydroxylation of multiple conserved proline and lysine residues and glycosylation 

of hydroxylysines. 

BIOLOGICAL ACTIVITIES OF ADIPONECTIN MULTIMERS 

Discussion of the biological activities of the different adiponectin multimers has 

proven controversial. HMW multimer levels appear to be higher in women compared 

to men 26 and adiponectin exerts multiple metabolic actions at multiple tissue 

sites. The isolated globular domain of adiponectin stimulates fatty acid oxidation 

in skeletal muscle, whereas full length adiponectin synergizes with insulin to 

inhibit hepatic glucose production. 16,29,30 In mice, disruption of the adiponectin 

locus leading to its ablation resulted in impaired fatty acid clearance, increased 

tumor necrosis factor-α levels, and aggravated insulin resistance in animals fed 

high fat diets. 31,32 

The HMW and hexameric adiponectin can activate transcription factor NFκB 

in undifferentiated or differentiated C2C12 cells, but trimeric adiponectin and 

the isolated globular domain of adiponectin cannot. The isolated globular domain 

of adiponectin, but not full-length adiponectin hexamer, enhances muscle fatty 

acid oxidation by inactivating acetyl-CoA carboxylase following stimulation of 

AMP-activated protein kinase (AMPK). 33,34 Waki et al. reported specifically that 

the HMW isoform promotes AMP-activated protein kinase in hepatocytes. 26 In 

contrast, Tsao et al. recently reported that only trimers activate AMPK in muscle, 

whereas hexamers and HMW form activated NF-κB. 35 

Differences in the tissue-specific expression patterns of two adiponectin 

receptors may contribute to these divergent activities. 36 Bobbert et al. investigated 

the effects of moderate weight reduction by a lifestyle intervention on adiponectin 

oligomer composition and its relation to glucose and fat metabolism. 37 While 

HMW and MMW adiponectins increased, a decreased amount of LMW adiponectin 

was found after weight reduction. Total adiponectin and especially HMW 

adiponectin correlated strongly with HDL cholesterol while correlations with 

other markers of glucose or fat metabolism were weak. 

Many studies have demonstrated a correlation between total adiponectin and 

HDL cholesterol. Most studies suggested that hypoadiponectinemia is more 

closely related to adiposity and dyslipidemia rather than insulin sensitivity. 38 

Indeed, Bobbert et al. found a strong correlation between HMW adiponectin and 

HDL cholesterol, which suggests that the relationship between total adiponectin 

and HDL cholesterol is primarily driven by HMW adiponectin rather than total 

adiponectin (Figure 10.1). 

HDL cholesterol is basically generated from lipid-free apolipoprotein A-I or 

lipid-poor pre-ß1-HDL as a precursor. These precursors are partially produced 

by the liver and it is well known that adiponectin oligomers specifically affect 

liver metabolism. 25 The importance of considering adiponectin oligomers is supported 

by a multivariate analysis revealing that HMW adiponectin explained about 

30% of the variability of HDL cholesterol (Figure 10.2). These results confirm 

those of Baratta and co-workers, who demonstrated that adiponectin is correlated


a) 

b) 

HDL (mmol/l) 

HDL (mmol/l) 

2.0 

1.5 

1.0 

0.5 

0.0 

2.0 

1.5 

1.0 

0.5 

Adiponectin (μg/ml) 

r = 0.61 (p = 0.016) 

0 2 4 6 8 10 12 

r = 0.665 (p = 0.007) 

0.0 

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 

HMW absolute (μg/ml) 

FIGURE 10.1 Correlation between (a) total adiponectin and HDL and (b) absolute concentrations 

of adiponectin oligomers and HDL in participants of a weight reduction 

program after moderate weight loss.


with serum lipid improvement independently of insulin sensitivity changes after 

weight loss. 39 

p38MAP kinase 

Glc uptake 

Skeletal muscle 

AdipoR1 AdipoR2 

P CBS 

α γ 

AMPK β 

FA oxidation 

Skeletal muscle 

FIGURE 10.2 Adiponectin-dependent intracellular pathways. 

Different adiponectin oligomers and the varying appearances of the AdipoR1 

and AdipoR2 adiponectin receptors may be responsible for the different actions 

of adiponectin oligomers on fat or glucose metabolism. However, the precise 

molecular mechanism for the oligomer-specific effect is still unclear. The mechanism 

that regulates adiponectin oligomer composition has not been identified. 

Data from intervention studies with thiazolidinediones suggest that the process 

is PPAR-γ-dependent. 40 Because PPAR-γ is activated by negative energy balance 

and weight reduction, 41 the effect on adiponectin oligomers may also depend on 

PPAR-γ-related pathways. Tsuchida et al. showed the influence of PPAR-α and 

γ on the expression of adiponectin and AdipoR1 and AdipoR2. They showed that 

activation of PPAR-γ or food restriction increased the ratio of HMW to total 

adiponectin and that activation of PPARα did not affect the ratio. 

RESULTS OF WEIGHT LOSS 

PPARα 

FA oxidation 

Liver 

The results of Bobbert et al. are in agreement with other studies showing that 

moderate weight loss results in relatively small changes of circulating adiponectin 

levels. 42 One study of six patients and a 3-month follow-up investigated the effects


of weight reduction on adiponectin oligomers. This study primarily aimed to 

investigate the effects of HMW adiponectin on endothelial cell apoptosis. Due 

to the small number of participants, the precise relationship of adiponectin oligomers 

to metabolic changes after weight loss was not further evaluated. 43 

Physical activity correlates strongly with obesity and is apart from dietary 

interventions a major part of weight reduction. Physical exercise is associated 

with reduced risks for the development of obesity-associated co-morbidities like 

type 2 diabetes mellitus 44 and also reduces the mortality risks of individuals with 

impaired glucose tolerance to the levels of healthy persons. 45 The improvement 

of insulin sensitivity by physical activity has been proposed as a possible mechanism 

of these effects. 46 From a mechanistic view, adipokines have been identified 

as potential mediators between obesity and insulin sensitivity. Despite the temptation 

to speculate that circulating adiponectin may be affected by degrees of 

physical activity, most studies have found that physical exercise has no influence 

on total adiponectin levels. Neither short term exercise nor long term physical 

training exerted effects on total adiponectin plasma levels. 47,48 Rather controversially, 

a study by Jurimae et al. reported reductions directly after acute rowing. 

However, 30 min after the rowing levels increased above resting values, 49,50 

Kriketos et al. observed increased adiponectin levels following two or three bouts 

of exercise over 1 week. These values remained elevated after 10 weeks of 

exercise. 49,50 Bobbert et al. showed that total adiponectin and oligomers were 

unchanged by acute and chronic exercise (in press). Total adiponectin levels and 

oligomers were not different for trained or untrained persons and were also 

unaltered by different training intensities. It is therefore unlikely that changes of 

adiponectin oligomers are responsible for the beneficial effects of sustained 

physical exercise on lipid and glucose metabolism. However, total adiponectin 

and again specifically HMW oligomers correlated with HDL cholesterol in this 

study, which supported the hypothesis that HMW adiponectin specifically mediates 

the positive effects of adiponectin. 

CENTRAL NERVOUS SYSTEM EFFECTS 

In addition to the peripheral influence of adiponectin on human metabolism, 

central nervous effects also play an important role in the regulation of human 

energy balance and metabolism. A growing body of evidence suggests that adiponectin 

directly affects energy balance by increasing thermogenesis. 51 Adiponectin 

has recently been reported to generate a negative energy balance by increasing 

energy expenditure. 52 

Spranger et al. showed that neither radiolabeled non-glycosylated nor glycosylated 

globular adiponectin crossed the blood–brain barrier (BBB) in mice. 53 

In addition, adiponectin and adiponectin oligomers were not detectable in human 

cerebrospinal fluid using various established methods. Using murine cerebral 

microvessels, they demonstrated expression of adiponectin receptors that were 

up-regulated during fasting in brain endothelium. Interestingly, treatment with 

adiponectin reduced secretion of centrally active interleukin-6 from brain


endothelial cells — a phenomenon paralleled by similar trends of other 

pro-inflammatory cytokines. These data suggest that direct effects of endogenous 

adiponectin on central nervous system pathways are unlikely to exist. However, 

the identification of adiponectin receptors on brain endothelial cells and the 

finding of a modified secretion pattern of centrally active substances from BBB 

cells provide an alternate explanation as to how adiponectin may evoke effects 

on energy metabolism. 

SUMMARY 

Adiponectin plays an important role in human metabolism, although the exact 

function of adiponectin is still unclear. The influences of adiponectin and adiponectin 

oligomers on peripheral glucose and lipid metabolism serve as foci of 

current clinical research. Some in vitro analyses showed first hints of adiponectin 

oligomer-dependent intracellular signalling cascades and initial human data demonstrated 

the different peripheral and central nervous system actions of total 

adiponectin and the different adiponectin oligomers. 

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11 Insulin-Stimulated 

Reactive Oxygen 

Species and Insulin 

Signal Transduction 

CONTENTS 

Barry J. Goldstein, Kalyankar Mahadev, and 

Xiangdong Wu 

Introduction .......................................................................................................178 

Regulation of Reversible Tyrosine Phosphorylation in Insulin 

Signaling by Tyrosine Phosphatases .......................................................178 

ROS as Second Messengers for Cellular Tyrosine Kinase Signaling .............179 

Novel Regulatory Paradigm: PTPs Are Thiol-Dependent Enzymes 

Regulated by Cellular ROS.....................................................................180 

Generation of H 2O 2 by Cellular Insulin Stimulation .......................................181 

Insulin-Stimulated H 2O 2 Generation Negatively Regulates PTP1B ................182 

Dynamics of Receptor-Induced ROS, PTP Inhibition, and 

Signal Transduction.................................................................................183 

Identification of the NADPH Oxidase Homolog Nox4 as a Potential 

Mediator of Insulin-Stimulated ROS ......................................................184 

Regulation of Nox4 Signaling in Insulin Action Cascade...............................184 

Potential Role of Rac in Insulin-Stimulated H 2O 2 and PTP Regulation .........185 

Gαi2.........................................................................................................185 

Novel Targets of Insulin-Stimulated ROS That May Potentially 

Influence Insulin Action ..........................................................................186 

Summary ...........................................................................................................186 

Acknowledgment...............................................................................................187 

References .........................................................................................................187 

177


INTRODUCTION 

Cellular reactive oxygen species (ROS; superoxide and H 2O 2), especially when 

chronically raised to high levels and associated with hyperglycemia, are widely 

recognized to play an important pathophysiological role in the chronic complications 

of diabetes as well as in the development of the disease. 1–3 In contrast, 

the transient generation of smaller amounts of ROS is triggered in cells in response 

to stimulation with a variety of growth factors, cytokines, and hormones including 

insulin, and facilitates their respective signaling cascades. The involvement of an 

oxidation step in the action of insulin has been suggested for decades, but only 

recently have potential molecular mechanisms been identified for these effects. 

Among the signaling enzymes potentially susceptible to inhibition by biochemical 

oxidation are those that contain reduced cysteine thiol side chains 

essential for their catalytic activities, including the family of protein–tyrosine 

phosphatases (PTPs) and other important signal regulators. Recently, we identified 

a role for the NADPH oxidase homolog known as Nox4 in the rapid generation 

of ROS in insulin-stimulated cells. 4–6 A full understanding of this signaling 

network may potentially provide a novel means of facilitating insulin action in 

states of insulin resistance and differentially regulating some of the pleiotropic 

cellular actions of insulin. 

REGULATION OF REVERSIBLE TYROSINE PHOSPHORYLATION 

IN INSULIN SIGNALING BY TYROSINE PHOSPHATASES 

Insulin is a critical regulator of pleiotropic cellular responses and resistance to 

the action of insulin in peripheral tissues is a fundamental defect in type 2 

diabetes. 7,8 Insulin action is initiated by binding to a specific plasma membrane 

receptor that encodes a tyrosine-specific protein kinase that autophosphorylates 

the receptor and its substrate proteins in cells. 9,10 These phosphorylated tyrosine 

motifs act as docking scaffolds for the binding and activation of a variety of 

signaling and adaptor proteins that are linked to downstream insulin responses. 

Specific PTPs regulate the steady-state balance of reversible protein–tyrosine 

phosphorylation in the insulin signaling cascade, in concert with the insulin 

receptor kinase. 5 In addition to serving as steady-state regulators, PTPs appear 

to be required for receptor deactivation since purified insulin receptors retain their 

autophosphorylation state after insulin is removed from the ligand binding site 

in vitro, and in vivo, dissociation of insulin from the receptor is followed by its 

rapid dephosphorylation and deactivation of its kinase activity. 11 

In particular, PTP1B has become an important target for therapeutic intervention 

in disease states associated with clinical insulin resistance. 12–14 This 

single-domain intracellular PTP has been convincingly shown in two knock-out 

mouse models to negatively regulate the insulin action cascade in vivo. 15,16 The 

cellular basis of these in vivo findings is well documented. 12 PTP1B is active in 

vitro against the autophosphorylated insulin receptor 17,18 and it also has a relatively 

high specific activity toward IRS-1 compared to other candidate PTPs. 19,20 Several

Oxygen Species and Signal Transduction 179 

studies have characterized the unique molecular interactions underlying the close 

interaction between the insulin receptor and PTP1B that is facilitated by the 

presence of a second phosphotyrosine binding site in the PTP1B catalytic region 

that interacts with the multiple phosphotyrosine residues of the receptor 

kinase. 21–23 

The PTPs comprise an extensive group of homologous proteins involved in 

a variety of signal transduction pathways. 24 Receptor and non-receptor forms of 

the classical tyrosine-specific PTPs have in common a ~230 amino acid phosphatase 

domain that contains the tightly conserved signature catalytic motif 

VHCSxGxGR[T/S]G. 25 The non-classical, dual-specificity (tyrosine and serine/ 

threonine) phosphatases (e.g., MAP kinase phosphatase and others) 26 and PTEN 

(phosphatase and tensin homolog deleted on chromosome 10), which dephosphorylates 

the 3-phosphate of inositol phospholipids generated by PI 3-kinase 

and exhibits dual phosphatase activity in vitro are structurally related, sharing a 

less tightly conserved catalytic motif that retains the essential C(x) 5R core structure. 

27 This sequence contains the reduced cysteine residue required for catalysis 

that is involved in the formation of a cysteinyl-phosphate intermediate. 28,29 Modification 

of this catalytic cysteine by oxidation or disulfide conjugation is a 

critically important mode of reversible and irreversible PTP regulation in vivo, 

including in the insulin action cascade. 30–32 

ROS AS SECOND MESSENGERS FOR CELLULAR TYROSINE 

KINASE SIGNALING 

Superoxide and H 2O 2 are now well recognized to play an integral role in several 

growth factor and cytokine signal transduction pathways (recently reviewed in 

Rhee). 33 Superoxide [O 2• – ], hydroxyl [•OH] ions, and H 2O 2 generated by cellular 

redox reactions have complex physiologies and are ultimately converted to H 2O 

+ O 2 by cellular catalase, thioredoxin, glutathione peroxidase, and/or peroxiredoxins. 

Relatively low levels of H 2O 2 generated in response to growth factor stimulation 

occur in a concerted fashion with specific signaling targets in cells, suggesting 

a role as a second messenger. H 2O 2 activates tyrosine phosphorylation 

cascades in cultured cells in a manner that mimics ligand-mediated signaling by 

PDGF and EGF. However, intracellular H 2O 2 generated transiently during stimulation 

of cells with growth factors has been convincingly demonstrated in seminal 

experiments by the groups of Finkel et al. 34 and Rhee et al., 35 showing that 

autophosphorylation of PDGF and EGF receptors, respectively, and their distal 

signaling effects are dependent on post-receptor intracellular H 2O 2 production, 

not simply the addition of exogenous H 2O 2. As described below, numerous studies 

support the hypothesis that cellular oxidant signaling is mediated by discrete, 

localized redox circuitry, distinct from the notion of a generalized “oxidative 

stress” effect. 36


NOVEL REGULATORY PARADIGM: PTPS ARE THIOL-DEPENDENT 

ENZYMES REGULATED BY CELLULAR ROS 

In parallel with developments in the cellular physiology of H 2O 2 generation, 

studies have also characterized the biochemical inhibition of PTPs by progressive 

oxidation of their catalytic cysteine thiol moieties by cellular ROS to more inert 

forms in vivo (Figure 11.1). 30,32,37–39 The activity of PTP1B is dependent on the 

oxidation state of its cys-215 residue, which is required for catalytic activity via 

the formation of a phospho-enzyme intermediate. 29,37,40,41 

The catalytic cysteine residue in the PTP active site is particularly sensitive 

to oxidation because of hydrogen bonding of neighboring side chains, which 

lowers the thiol p Ka to ~5.5, more than 3 units below that of a typical –SH group, 

rendering it in an ionized state at physiological pH. Compared to other typical 

protein sulfhydryl side chains, the catalytic PTP thiol can be readily oxidized by 

PTP-SSG 

(glutathiolated) 

inactive 

GSH 

GSH ? 

GSH 

GSSG 

PTP-S- PTP-S- active 

O• 

PTP-SOH 

(sulfenic acid) O• 

inactive 

PTP-SN 

(sulfenyl-amide) 

inactive 

PTP-S- PTP-S- reactivated 

thiol 

reduction 

intramolecular 

rearrangement 

thiol 

reduction 

PTP-SO 2 H PTP-SO 3 H 

inactive, irreversible 

FIGURE 11.1 Regulation of PTP catalytic activity by oxidation, reduction, and conjugation. 

The catalytic cysteine residue of PTPs is especially reactive because of the low p Ka 

of the sulfhydryl that favors a relatively ionized state of the cysteinyl hydrogen. 42 When 

subjected to ROS, including those elicited by cellular insulin stimulation, the cysteine side 

chain undergoes step-wise oxidation to increasingly inert forms. 32,38,106 The inactive 

sulfenic derivative may be reduced to regenerate the active thiol form of the protein. 

Alternatively, it may be directly conjugated with glutathione in the cell, producing a 

catalytically inert PTP derivative that can be reactivated by biochemical reduction or 

through the action of glutathione reductases. 46 Recently, the sulfenic derivative of PTP1B 

has been shown to undergo an intramolecular rearrangement, forming a novel sulfenyl–amide 

derivative that also sequesters the PTP in an inactive state. 47,48 The sulfenyl–amide 

form may actually be an obligate intermediate in this reaction scheme because 

its altered protein conformation opens a groove adjacent to the catalytic center that may 

render it particularly susceptible to reduction with cytosolic glutathione compared with 

the sulfenic derivative. (Reprinted from Goldstein, B.J. et al. Diabetes 54, 311, 2005. With 

permission from the American Diabetes Association.)


locally generated H 2O 2 even in the presence of high cytosolic concentrations 

(millimolar) of the cysteine-containing tripeptide glutathione (GSH). 42 

The catalytic cysteine thiol is initially oxidized to the sulfenic (–SOH) form 

that can be reversed by cellular enzymatic mechanisms or with reducing agents 

in vitro (Figure 11.1). 40,43 Sequential steps of progressive oxidation to the sulfinic 

(–SO 2H) and sulfonic (–SO 3H) forms can lead to irreversible PTP inactivation. 

41,44,45 However, the partially oxidized sulfenic acid intermediate of PTP1B 

can also be rapidly converted to other forms that may stabilize the molecule and 

protect it from further irreversible oxidation. 

One potentially stabilizing modification is conjugation with glutathione which 

may be enzymatically reactivated in cells by glutaredoxin. 46 The catalytic cysteine 

of PTP1B has recently been shown to be reversibly converted to a previously 

unknown intramolecular sulfenyl–amide species, in which it becomes linked to 

the main chain nitrogen of an adjacent residue, rendering the enzyme inactive 

and inducing large conformational changes that inhibit substrate binding. 47,48 This 

novel protein modification not only protects the enzyme catalytic site from irreversible 

oxidation to sulfonic acid but also permits redox regulation of the enzyme 

by promoting its reversible reduction by thiols. The conformation of the catalytic 

cleft assumes a more open structure in the sulfenyl–amide derivative of PTP1B 

and renders it particularly amenable to reduction by GSH. 47,48 This suggests that 

this unique protein derivative may, in fact, be an obligatory intermediate in the 

generation of the glutathionylated form of the oxidized enzyme. 

GENERATION OF H 2O 2 BY CELLULAR INSULIN STIMULATION 

The potential involvement of oxidant species in insulin signaling was initially 

explored more than 30 years ago, with the observation by Czech et al. that certain 

metal cations interacting with albumin could transfer electrons to a cellular target 

and enhance glucose utilization by adipocytes. 49–51 Livingston et al. also showed 

that polyamines and related insulin mimickers acted via the generation of H 2O 2 52 

and that H 2O 2 stimulates lipid synthesis in adipocytes. 53 In complementary studies, 

insulin was also shown to stimulate the generation of H 2O 2 in adipocytes. 54 

An early characterization of the enzymology of this process revealed that 

insulin activated a plasma membrane enzyme system with the properties of an 

NADPH oxidase, resulting in the downstream production of H 2O 2. 55,56 Further 

biochemical studies of this activity showed that it accounted for insulin-stimulated 

ROS production in rat adipocyte plasma membranes 57,58 and was also present in 

3T3-L1 adipocytes. 59 NADPH oxidase catalyzes the reduction of oxygen to superoxide 

radical: 2 O 2 + NADPH → 2 O 2 •– + NADP + + H + . While superoxide anions 

can react with thiols, they are rapidly converted spontaneously or by superoxide 

dismutase in the cell to generate H 2O 2. 42


INSULIN-STIMULATED H 2O 2 GENERATION NEGATIVELY 

REGULATES PTP1B 

One hypothesis drawn from the diverse research data described above suggests 

that plasma membrane oxidase activity stimulated by insulin generates cellular 

ROS which, in turn, facilitates the insulin signaling cascade via the oxidative 

inhibition of cellular PTP activity, in particular involving PTP1B. 5,6 We reported 

that in the 3T3-L1 adipocyte cell model, insulin stimulated the generation of 

cellular ROS in minutes within the high physiologic range of insulin concentrations. 

30,31 

Blocking insulin-stimulated ROS with diphenyleneiodonium (DPI), an inhibitor 

of cellular NADPH oxidase activity, or catalase, reduced the insulin-stimulated 

autophosphorylation of the insulin receptor and the IRS proteins consistent 

with the notion that the oxidant signal inhibited cellular PTPs that serve as 

negative regulators of the insulin signaling cascade. That the enhancement of 

insulin signaling by the oxidant signal was associated with PTP inhibition was 

also shown by a novel approach that includes sample handling and analysis under 

anaerobic conditions to preserve the endogenous activity of PTPs isolated from 

cultured cells and avoiding cysteine oxidation that occurs on exposure to air. 60 

In HepG2 hepatoma cells, stimulation with 100 nM insulin for 5 min reduced 

overall PTP activity in the cell homogenate, and biochemical reduction of the 

enzyme samples with dithiothreitol prior to PTP assay fully restored the reduced 

PTP activity of the insulin-treated samples, indicating that they had been reversibly 

oxidized and inactivated by insulin exposure. 30 

Similar effects were observed in 3T3-L1 adipocytes. Insulin-stimulated generation 

of H 2O 2 also affected the specific activity of endogenous PTP1B isolated 

from intact cells. In 3T3-L1 adipocytes, insulin treatment also potently reduced 

the activity of immunoprecipitated PTP1B that was also reversible toward control 

levels by preincubation with dithiothreitol prior to assay. 30 In the continued 

presence of insulin, this effect was sustained for at least 10 min. Catalase pretreatment 

of the cells abolished the insulin-induced inhibition of PTP1B. Oxidative 

inactivation of PTP1B is thus associated with enhanced insulin signal transduction 

via the insulin-stimulated H 2O 2 signal. 

Further support of the notion of ROS serving as an enhancer of insulin action 

was provided by McClung and colleagues 61 who generated a line of mice overexpressing 

cellular glutathione peroxidase and showed that they developed hyperinsulinemia 

and hyperglycemia with insulin resistance and obesity. Insulin signaling 

was diminished in the tissues with reduced insulin-stimulated insulin 

receptor β-subunit phosphorylation and activation of Akt. Since excess glutathione 

peroxidase quenches intracellular ROS generation, it was hypothesized 

to interfere with insulin action by blocking PTP inactivation. Another group has 

also reported that an inability to generate ROS in response to insulin stimulation 

accounts for insulin insensitivity of ERK activation in SK-N-BE(2) neuroblastoma 

cells. 62


DYNAMICS OF RECEPTOR-INDUCED ROS, PTP INHIBITION, 

AND SIGNAL TRANSDUCTION 

ROS production following ligand stimulation, including insulin, generates only 

a fraction of the ROS concentration observed in phagocytic cells and follows a 

brief time course, on the order of minutes. 30 These features apparently account 

for the signaling role of insulin-induced ROS compared to the chronic exposure 

to ROS in patients with hyperglycemia that is associated with organ dysfunction 

and chronic complications of diabetes mellitus. 63 

The low levels of ROS in insulin signaling imply specific cellular protein 

targets that are particularly susceptible to oxidative modification. This biochemical 

evidence, therefore, also supports the notion of a discrete network of “redox 

circuitry” 42,64,65 with temporal and spatial influences that are likely to correspond 

to other regulatory aspects of the insulin action pathway. 66 Work by Stone and 

colleagues 36 clearly describes the gradient of cellular responses to oxidative 

stimulation, with low levels eliciting signaling responses and cell proliferation 

without cellular oxidation of most free thiols such as glutathione. Increasing 

exposure to ROS is followed by growth arrest, cellular damage, thiol oxidation, 

and induced cell death. 

Insight into the disposition of growth factor-induced ROS has recently been 

gleaned from elegant studies by Reynolds et al. who modeled EGF receptor 

activation and signal propagation with PTP inhibition by ROS. 67 ROS generated 

in response to EGF stimulation in MCF7 cells were spatially constrained to a 

layer below the plasma membrane. 

Using reaction constants gleaned from published experimental work, including 

PTP inhibition by H 2O 2 and related effects, a model was developed that was 

most consistent with a bistable activation state for PTP and receptor tyrosine 

kinase activity. The formation of a reaction “wavefront” is postulated to involve 

local cycles of EGFR activation, H 2O 2 production, and PTP inhibition, which 

propagates along the plasma membrane. The model proposes that signal initiation 

involving oxidative inhibition of PTPs adds a feedback control loop to a reaction 

network that responds in an amplified and switch-like manner, especially at low 

levels of ligand stimulus. 

Consistent with this model, they also showed experimentally that blocking 

ligand-dependent H 2O 2 generation with the NADPH oxidase inhibitor DPI abolished 

the propagation of receptor phosphorylation and the amplification of receptor 

activation at low concentrations of EGF, converting the system to a “stable” 

steady state by generating a more linear phosphorylation response to ligand 

stimulation. 67 Diminishing PTP inactivation with DPI also suppresses insulin 

receptor activation and several aspects of the downstream insulin signaling cascade. 

31,68 Thus, it would be of interest to employ these types of novel imaging 

techniques to evaluate the role of similar regulatory networks in insulin-sensitive 

cells.


IDENTIFICATION OF THE NADPH OXIDASE HOMOLOG NOX4 

AS A POTENTIAL MEDIATOR OF INSULIN-STIMULATED ROS 

Using the prototypic NADPH oxidase (Nox) catalytic gp91phox subunit, Cheng 

and Lambeth cloned a small family of five homologous Nox catalytic subunits 

(reviewed in Lambeth 69 ). Although Nox4 is prominently expressed in kidney, 70–72 

we also showed that it was expressed among insulin-sensitive cell types including 

liver, skeletal muscles, and adipocytes. 4 Evidence for an integral role of Nox4 in 

the insulin-induced oxidant signal was obtained by adenovirus-mediated expression 

of Nox4 deletion constructs lacking either the NADPH binding domain or 

the combined FAD/NADPH domains that acted in a dominant-negative fashion 

and attenuated insulin-stimulated generation of H 2O 2. 

Functionally, expression of the deletion constructs led to an inhibition of 

insulin receptor and IRS-1 tyrosine phosphorylation, activation of downstream 

serine kinases, and glucose uptake. Similar results were obtained with transfection 

of siRNA oligonucleotides that reduced Nox4 protein abundance. 4 Altogether, 

these results suggest that Nox4 overexpression potentiates, and reduced Nox4 

mass diminishes, insulin signal transduction in 3T3-L1 cells. 

ROS generation by Nox4 in adipocytes was also associated with oxidative 

inhibition of cellular PTP1B activity. 4 Overexpression of recombinant PTP1B 

inhibited insulin-stimulated tyrosine phosphorylation of the insulin receptor, 

which was significantly reversed by co-overexpression of active Nox4. The effect 

of overexpression of Nox4 on receptor autophosphorylation was closely associated 

with inhibition of PTP1B catalytic activity, measured in enzyme immunoprecipitates. 

REGULATION OF NOX4 SIGNALING IN INSULIN 

ACTION CASCADE 

Clarification of the regulation of Nox4 by growth factors has been elusive. 73 All 

the Nox catalytic subunits analogous to gp91phox have been shown to be physically 

associated with p22phox, an essential component of the flavocytochrome 

complex 74 ; thus, regulation of p22 abundance may be a means of regulating Nox4 

activity. The phagocyte NADPH oxidase has been extensively characterized; the 

regulatory proteins p47phox and p67phox have been shown to play a key role in 

protein complex formation with activation of Nox2. 69,75 

Since these proteins are not expressed in non-phagocytic cells, several groups 

have recently reported the cloning of related interacting proteins termed NOXO1 

(Nox organizer 1 or p41) and NOXA1 (Nox activator 1 or p51) that are homologs 

of the phagocyte p47phox and p67phox proteins. 76–79 Superoxide production by 

Nox1 and Nox3 is enhanced by interactions with these regulatory subunits. 80–83 

Nox5 is regulated by intracellular calcium levels. 84 NOXO1 and NOXA1 do not 

affect Nox4 activity and currently the mechanisms underlying the regulation of 

Nox4 activity are not known. NIH 3T3 fibroblasts transfected with Nox4 exhibit


constitutively increased superoxide generation, 70 suggesting that Nox4 may not 

depend on activation by regulatory subunits. However, the mechanism of the rapid 

activation of Nox4 in insulin-stimulated ROS generation remains unexplained. 

POTENTIAL ROLE OF RAC IN INSULIN-STIMULATED H 2O 2 

AND PTP REGULATION 

Rac is a key component of the NADPH oxidase complex in a variety of cell 

types. 73,85 A chimeric Rac1-p67phox protein increases Nox2 activity, expression 

of a dominant-negative Rac inhibits the rise in ROS seen after stimulation by 

growth factors or cytokines, and a constitutively active Rac stimulates ROS 

formation in NIH 3T3 cells and in renal mesangial cells. 86,87 Since superoxide 

generation in the renal cell system is likely to involve Nox4, these data also 

implicated Rac as a potential regulator of this Nox homolog. 87 

In insulin-sensitive cells, Rac is involved in distal insulin signaling to glucose 

transport. 88 However, to date, our experiments using overexpression of wild-type, 

dominant-negative, and constitutively active Rac in differentiated 3T3-L1 adipocytes 

have not demonstrated a role for Rac in insulin-stimulated receptor autophosphorylation 

or substrate (IRS-1) tyrosine phosphorylation. 

GαI2 

Data from a variety of sources over the years have also linked the small G-protein 

Gαi2 with insulin action and potentially with insulin-stimulated NADPH oxidase 

activity (reviewed by Waters et al. and Malbon 89,90 ). Insulin-stimulated plasma 

membrane NADPH oxidase was shown to be coupled to Gαi2 91 and more recently 

insulin stimulation led to protein association between Gαi2 and the insulin receptor. 

92 Insulin receptor autophosphorylation was stimulated by activated Gαi2, and 

blocked by pretreatment with pertussis toxin, consistent with an earlier paper on 

Fao hepatoma cells. 93 

A recent paper also linked the attenuation of platelet activation by insulin 

with tyrosine phosphorylation of Gαi2 and complex formation between IRS-1 

and Gαi2, but not other Gα subunits. 94 Other approaches including a series of 

studies by Malbon and colleagues in transgenic mice have supported a permissive 

role of Gαi2 in insulin signaling in vivo. 95–98 Interestingly, mice lacking Gαi2 

also exhibited increased tissue PTP activity, 95 implicating a potential loss of 

insulin-stimulated NADPH oxidase activity in Gαi2-deficient animals with 

reduced oxidative inhibition of PTPs that regulate the insulin action pathway. 

Overall, these studies are consistent with the hypothesis that the regulation of 

tyrosine phosphorylation in the insulin signal cascade is propagated by a wave 

of H 2O 2, possibly generated by a link between the insulin receptor and Gαi2, 

coupled to cellular NADPH oxidase activity, which transiently inhibits PTP 

activities.


NOVEL TARGETS OF INSULIN-STIMULATED ROS THAT MAY 

POTENTIALLY INFLUENCE INSULIN ACTION 

In addition to PTPs that have been implicated in the regulation of insulin signaling 

(e.g., PTP1B and LAR, as discussed above 11 ), a number of additional cellular 

enzymes are potential targets of oxidative inhibition by insulin-induced ROS. The 

serine–threonine phosphatase PP2A implicated in the negative regulation of Akt 

by dephosphorylation of ser-473 has a redox-sensitive cysteine residue that is 

potentially susceptible to inhibition by H 2O 2. 99 

The dual-specificity (ser/thr and tyr) phosphatase MKP-1, which attenuates 

insulin-stimulated MAP kinase activity, 100 is also dependent on a reduced thiol 

for activity. The lipid phosphatase PTEN can modulate downstream insulin signaling, 

101 and is also inactivated by oxidation of essential cysteine residues in its 

active site, which can be reactivated by thioredoxin in cells. 102,103 Further research 

is needed to determine the effects of the oxidative inhibition of these important 

signaling regulators on proximal and distal events in the insulin action cascade. 

Rhee and his group have shown that even in a cellular milieu containing 

millimolar concentrations of slowly reactive thiols like GSH, only a limited set 

of proteins are rapidly oxidized by growth factor-stimulated ROS, including 

PTP1B and a few other proteins with reactive cysteines including protein disulfide 

isomerases, thioredoxin reductase, and creatine kinase. 104,105 We have confirmed 

similar findings following cellular insulin stimulation of insulin target cell types 

(Wu et al., unpublished data), and are currently employing novel fluorescently 

tagged thiol reagents (iodoacetamide and maleimide derivatives) and adapting 

proteomic methods for the differential labeling of protein thiol before and after 

cellular insulin stimulation. 

Further work will help define the regulatory components and mechanism of 

NADPH oxidase activation by insulin in various insulin-sensitive cell types and 

the effects of insulin-stimulated ROS on the insulin action cascade with the 

identification of specific cellular targets susceptible to oxidative modification by 

insulin-stimulated ROS. Elucidation of these processes will determine how they 

are involved in the normal physiology of insulin signaling, whether they contribute 

to insulin-resistant disease states, and whether elements of this system may 

emerge as novel targets for pharmaceutical intervention. 

SUMMARY 

Reversible tyrosine phosphorylation plays an essential role in the regulation of 

transmission of the insulin signal at receptor and post-receptor sites in the insulin 

action pathway. PTPs, in particular PTP1B, and other thiol-sensitive signaling 

proteins are integral to the negative regulation of insulin signaling. A growing 

body of data over the past three decades has led to the appreciation that cellular 

stimulation with insulin generates ROS that can inhibit these negative regulators 

by oxidative biochemical alterations which, in turn, can facilitate the insulin 

signaling cascade. With the recognition that a small family of NADPH oxidase


α-Subunit 

β-Subunit 

FIGURE 11.2 Insulin-induced ROS production and PTP regulation via Nox4 in insulinsensitive 

cells. This figure illustrates the action of insulin to stimulate receptor tyrosine 

autophosphorylation which activates the receptor toward its cellular substrate (IRS) proteins. 

The receptor and IRS tyrosine phosphorylation require cellular PTP activity to return 

to a basal state. The insulin receptor is coupled to the Nox4 homolog of NADPH oxidase 

and stimulates the cellular generation of reactive oxygen species which, in turn, leads to 

oxidative inhibition of the thiol-dependent PTPs, including PTP1B, the major phosphatase 

for the insulin signaling cascade. The lower right portion illustrates the PTP reaction 

mechanism in which the reduced thiol side chain in the enzyme catalytic domain forms 

a phosphocysteine intermediate with the phosphotyrosine substrate. If the catalytic PTP 

thiol is oxidized, this reaction intermediate cannot form and the enzymatic reaction is 

blocked. See text for discussion and references. 

homologs catalyzes the generation of ROS at the plasma membrane, we have 

recently provided evidence in the 3T3-L1 adipocyte system that Nox4, which is 

expressed in insulin-sensitive cell types, is a novel molecular target that may 

mediate this process (Figure 11.2). 


This work was supported by National Institutes of Health grant RO1 DK43396 

to Dr. Goldstein. 

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Insulin 

ATP 

-pY 

-pY 

-pY 

Y - Substrate Substrate - pY 

-PO 4 

PTP1B 

-PO 4 

PTP Cys 

215 

PTP thiol 

NADPH 

Oxidase 

(Nox4) 

pY protein 

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12 

CONTENTS 

Intracellular Signaling 

Pathways and 

Peroxisome Proliferator- 

Activated Receptors in 

Vascular Health in 

Hypertension and in 

Diabetes 

Farhad Amiri, Karim Benkirane, and 

Ernesto L. Schiffrin 

Introduction .......................................................................................................196 

MAPK Signaling in Vascular Remodeling.......................................................198 

ERK1 and ERK2 Signaling, Effects of PPAR-γ, and Vascular Remodeling ..199 

p38 Signaling in Vascular Remodeling ............................................................200 

Role of JNK Signaling in Vascular Remodeling..............................................201 

PI3K Signaling Pathway and Vascular Remodeling ........................................201 

Role of Akt/PKB Signaling Pathway in Vascular Remodeling .......................202 

ROS and Vascular Remodeling.........................................................................202 

Vascular Inflammation and Remodeling...........................................................203 

Dual PPAR Activators.......................................................................................204 

Conclusion.........................................................................................................204 

References .........................................................................................................205 

195


INTRODUCTION 

In both hypertension and diabetes mellitus significant changes that occur in the 

vasculature affect both large and small arteries and lead to cardiovascular events 

such as myocardial infarction, stroke, peripheral vascular disease, and compromise 

of renal function. In addition, diabetes also involves microvascular disease 

in different vascular beds including the retina and the kidney that contribute to 

the morbidity, and in the case of the kidney, the mortality associated with diabetes. 

The degree and importance of vascular disease and its contribution to mortality 

in diabetes are such that diabetes has been called a vascular disease although 

its origin is undoubtedly metabolic. Moreover, hypertension and diabetes are 

often associated: approximately 20% of hypertensives may become diabetic and 

80 to 90% of type 2 diabetic patients develop hypertension. Description of the 

nature of vascular disease in hypertension and diabetes, its mechanisms, and 

therapeutic targets, potential and already demonstrated, become accordingly 

extremely important. Some of these will be dealt with in this chapter, particularly 

in relation to signaling pathways and putative vascular protective effects of 

activation of peroxisome proliferator-activated receptors (PPARs). 

Vascular injuries in large arteries in hypertension and diabetes differ mainly 

in intensity and are much more severe in diabetes. In large arteries, injuries of 

both the intima with development of atherosclerosis and of the media with 

arteriosclerosis occur. The description of the atherosclerotic process is beyond 

the scope of this chapter. It is sufficient to say that the severity of atherosclerosis 

in diabetes is such that it affects the peripheral circulation, leading to amputation 

of lower limbs, and also affects the coronary circulation where diabetes is considered 

a “coronary equivalent.” 1 

In small (resistance) arteries that measure 150 to 300 µm in lumen diameter, 

hypertension is associated with remodeling changes that lead to increased peripheral 

resistance — the hallmark of essential hypertension. The remodeling of small 

arteries in hypertension is eutrophic — reduced outer and lumen diameters with 

increased media-to-lumen ratio and no significant increase in cross-sectional area 

of the media. 2–4 Associated with the structure changes are deposition in the media 

of extracellular matrix components such as collagen and fibronectin 5 and dysfunction 

of the endothelium. 6 Diabetes, on the other hand, involves hypertrophic 

remodeling in which the media-to-lumen ratio is also increased along with an 

increased media cross-section, achieving true media hypertrophy. 7,8 Deposition 

of collagen is less important. More frequently than in hypertension, endothelial 

dysfunction is the norm. 8,9 

In the United Kingdom Prospective Diabetes Study (UKPDS), tight blood 

pressure (BP) controls in hypertensive patients with type 2 diabetes reduced the 

risk of macrovascular disease, stroke, and deaths related to diabetes. 10 Most 

hypertension randomized clinical trials failed to show beneficial effects on cardiac 

ischemia expected from population studies. Thus, blood pressure lowering may 

not be enough to normalize remodeled arteries in hypertensive or diabetic subjects. 

In hypertensive patients, the extent and consequences of tissue ischemia

Receptors in Vascular Health in Hypertension and Diabetes 197 

(in the heart, kidney, or brain) are influenced by small vessel disease. 3 The 

intermediate coronary lesions that are frequent in hypertensive and diabetic subjects 

will produce changes in flow if small artery remodeling is present. 

The renin–angiotensin–aldosterone system (RAAS) plays a critical role in 

the initiation and progression of cardiovascular disease. 11 The RAAS contributes 

to vascular remodeling of small arteries through different pathways. Angiotensin 

II (Ang II) can indirectly promote vascular remodeling through hemodynamic 

effects or directly activate myriad intracellular signaling pathways such as mitogen-activated 

protein kinase (MAPK), phosphoinositide-3 kinase (PI3K), Janus 

kinase/signal transducers and activators of transcription (JAK/STAT), and reactive 

oxygen species (ROS) through AT 1 receptors. Ang II may also transactivate 

growth factor receptors such as epidermal growth factor receptor (EGFR). 12 These 

pathways in turn initiate cascades in which different key proteins are stimulated, 

including nuclear factors that are responsible for cardiovascular gene transcription. 

The involvement of RAAS in hypertension and diabetes-related complications 

has been clarified through the use of a variety of RAAS inhibitors including 

but not limited to angiotensin converting enzyme (ACE) inhibitors, Ang receptor 

blockers (ARB), and aldosterone receptor antagonists. 3 

Insulin-sensitizing thiazolidinediones (TZDs) or glitazones may exert protective 

cardiovascular properties in part through the prevention of vascular remodeling. 

13 For instance, TZDs such as pioglitazone and rosiglitazone reduced blood 

pressure in several hypertensive rodent models including Ang II-infused rats, 

stroke-prone spontaneously hypertensive rats (SHRSP), and deoxycorticosterone 

acetate (DOCA)-salt hypertensive rats. 14–16 Blood pressure lowering was accompanied 

by prevention of vascular remodeling and endothelial dysfunction and 

down-regulation of inflammatory mediators in these rodent models. Although the 

hypotensive effects of TZDs observed in rodents were not observed in humans, 

other beneficial effects such as prevention of vascular remodeling, improvement 

of endothelial function, 17,18 and down-regulation of inflammatory markers 19 have 

been reported. 

TZDs mediate their effects through binding of PPAR-γ, a ligand-activated 

transcription factor belonging to the nuclear receptor superfamily. Forming a 

dimer with RXR and associated to co-activators and co-repressors, PPAR-γ binds 

to DNA PPAR response elements (PPREs) and regulates many genes implicated 

in carbohydrate metabolism, inflammation and thrombosis, endothelial function 

and cell growth. 20 Other PPAR isoforms include PPAR-α, which is activated by 

fatty acids and by lipid lowering fibrates and predominantly expressed in tissues 

exhibiting high fatty acid catabolism such as liver, heart, kidney, and skeletal 

muscle. Another isoform is PPAR-β/δ, which is expressed ubiquitously and is 

involved in fatty acid oxidation. 21 

The major beneficial effects of TZDs have been attributed to their actions on 

several protein kinases such has MAPK, PI3K, and ROS-generating enzymes, all 

of which have been implicated in vascular remodeling in hypertension. 14,22


MAPK SIGNALING IN VASCULAR REMODELING 

MAPK signaling has been largely studied for processes such as hyperplasia and 

hypertrophy associated with cell growth, and pro-inflammatory pathways, all of 

which are found in hypertensive and diabetic vascular remodeling. These 

serine/threonine kinases are subdivided into six subfamilies: extracellular signalregulated 

kinases 1 and 2 (ERK1/2), p38, c-jun N-terminal protein kinase (JNK), 

ERK3, ERK5, and ERK6. 23 Due to elaborate cross-talk among these kinases, we 

will focus only on ERK1/2, p38 and JNK proteins because they are the most 

commonly studied (Figure 12.1). 

Activation of MAPK is associated with cell growth, programmed cell death 

(apoptosis), cell transformation and differentiation, and cell contractility. ERK1/2 

proteins can be activated by different growth factors whereas p38 and JNK 

proteins are generally activated by cytokines and cellular stress. 23 Ang II is a 

potent stimulator of MAPK pathways through various upstream second messenger 

systems. 12,24,25 

MAPK activation requires phosphorylation of threonine and tyrosine residues 

by other kinases such as mixed lineage kinases (MLKs), MAPK kinase, or 

mitogen extracellular-regulated kinase (MEK). MEKs are activated by 

serine/threonine MEK kinases (Raf-1, A-Raf and B-Raf), which are in turn 

activated by small protein G (Ras family) and other kinases. 26,27 Raf phosphorylation 

is influenced by different kinases such as c-Src, protein kinase C (PKC), 

protein kinase B (PKB), and p21 (rac/Cdc42)-activated protein kinase (PAK). 26 

Stress, cytokines, 

growth factors Growth factors 

MLKs MLKs Raf 

MEK3/6 MEK4/7 MEK1/2 

p38 JNK 

Apoptosis, cell growth and 

differentiation, inflammation 

ERK1/2 

Cell growth and 

differentiation 

PD98059 

Stimulus 

MEKK 

MEK 

MAPK 

Biological 

response 

FIGURE 12.1 Mitogen-activated protein kinase (MAPK) signaling pathways and their 

responses. MLK = mixed lineage kinase (generic MEK). MEK = MAPK kinase. MEKK 

= MAPK kinase kinase.


ERK1 AND ERK2 SIGNALING, EFFECTS OF PPAR-γ, AND 

VASCULAR REMODELING 

ERK1 and ERK2 are ubiquitous proteins highly expressed in different tissues of 

the cardiovascular system (blood vessels, kidneys, heart). 28 Additionally, differential 

regulation occurs in different cell types. ERK2 expression was significantly 

higher than ERK1 expression in immune cells, 26 but both enzymes are equally 

and highly expressed in endothelial cells and vascular smooth muscle cells 

(VSMCs). 29–31 Ang II through binding to AT 1 receptors activates signaling cascades 

such as the Src homology 2 domain (Shc), c-Src, growth factor receptorbound 

protein 2 (Grb2), Son of sevenless (Sos), Ras-GTP, and MEK1, which 

then activate ERK1/2 through phosphorylation. 32 

Several studies using the MEK specific inhibitor PD98059 demonstrated an 

important role of ERK1/2 in the development and the maintenance of hypertension. 

33,34 For instance, in hypertensive vascular remodeling, ERK1/2 activation 

led to activation of phospholipase A 2, cyclooxygenase (COX)-2, and p90 ribosomal 

S6 kinase (p90Rsk), along with translocation of nuclear receptors and reorganization 

of cytoskeletal proteins implicated in cell growth and migration. 26 

More specifically, once ERK1/2 is phosphorylated, it will translocate to the 

nucleus and activate transcription of cell cycle genes. 23,26 

In VSMCs, ERK1/2 phosphorylation induced p90Rsk activation leading to 

ribosomal S6 protein phosphorylation and protein synthesis. In VSMCs derived 

from mesenteric arteries, we demonstrated the stimulatory effect of Ang II on 

cell hypertrophy, proliferation, and contractility. 29 Ang II-induced ERK1/2 activity 

was inhibited by PPAR-γ activation in conduit vessels whereas no effect was 

observed in mesenteric resistance vessels. 14 Unlike results of in vivo studies, in 

vitro acute stimulation of mesenteric VSMCs with Ang II-induced ERK1/2 activation 

was abrogated by PPAR-γ pre-stimulation with rosiglitazone (Figure 

12.2). 14 Taken together, these results suggest that the extracellular environment 

and duration of stimulation (acute versus chronic) affect PPAR-γ-modulated 

changes in Ang II-induced ERK1/2 activation. 

ERK1/2 may on the other hand inhibit PPAR-γ activity. In vivo, insulin, which 

constitutively activates MEKK1, induced in a ligand-independent manner PPAR-γ 

phosphorylation which increased TZD-dependent PPAR-γ trans-activation properties. 

35 In contrast, both epidermal growth factor (EGF) and platelet derived 

growth factor (PDGF) reduced PPAR-γ transcriptional activity in a ligand-dependent 

manner in adipocytes contained in the vascular periadventitial fat that regulates 

vascular function in paracrine fashion and may play an important role in 

blood pressure regulation and vascular remodeling. 36 

Although mesenteric artery PPAR-γ activity was reduced in Ang II-infused 

rats, albeit in the absence of changes in PPAR-γ expression, treatment with 

rosiglitazone prevented these changes, suggesting a potent negative regulatory 

role of PPAR-γ activators. 14 Endogenous PPAR ligands such as linoleic and 

retinoic acid may stimulate ERK1/2 activity in adipocytes and aortic VSMCs, 

respectively, 37,38 whereas prostaglandin J2 (PGJ2) activated ERK1/2 through PI3K


Resistance Artery SMC Resistance 

Artery 

PD98059 

Ang II/AT 1 

MEK 

ERK1/2 

Transcription 

factors 

Cell growth 

Ang II/AT 1 

No effect on 

ERK1/2 

Rosiglitazone Rosiglitazone 


vascular remodeling 

independent of ERK1/2 

Conduit 

Artery 

Ang II/AT 1 

MEK 

ERK1/2 

Transcription 

factors 


vascular remodeling 

FIGURE 12.2 Angiotensin (Ang) II effects on extracellular-regulated kinase (ERK) 1 and 

2 activation in resistance and conduit arteries. 

signaling in aortic VSMCs. These data collectively demonstrate the countervailing 

effects of PPAR activators on MAPK signaling pathways implicated in cell growth 

and migration induced by vasoactive agents such as Ang II. 

p38 SIGNALING IN VASCULAR REMODELING 

p38 MAPK comprises six isoforms (p38α1 and α2, p38β1 and β2, p38δ, and 

p38γ): the α and β isoforms are ubiquitous; the δ isoform is expressed mainly in 

kidneys, lungs, and pancreas; and the γ isoform is highly expressed in skeletal 

muscle. 39 p38 MAPK can be activated by a variety of stimuli including cytokines, 

growth factors, osmotic shock, and different stressors. p38 MAPK has been 

implicated in several pathophysiological conditions such as atherosclerosis, 

hypertensive vascular remodeling, and ischemic cardiomyopathy. 26 

Ang II can stimulate vascular p38 MAPK, affecting inflammatory responses, 

apoptosis, cell growth inhibition, and contractility. Inhibition of p38 MAPK 

reduced these effects in aortic but not mesenteric VSMCs. 40–42 p38 MAPK crosstalks 

with other members of the MAPK family (including ERK1/2, MEK3, and 

MEK6; see Figure 12.1) and other signaling molecules (e.g., heat shock protein 

27), which is important in cytoskeletal reorganization implicated in cell migration 

and vascular remodeling. 26,39 p38 MAPK is also regulated by PPAR-α and one 

of its co-activators, PGC-1, suggesting a regulatory role for p38 MAPK in PPARα-mediated 

lipid metabolism. 27 However, p38 MAPK does not seem to participate 

in PPAR-γ regulation, as inhibition of p38 MAPK failed to modulate PPAR-γinduced 

AT 1 gene transcription. 43 

This further demonstrates differential effects of different MAPKs on the RAAS. 

Since p38 MAPK may also induce VSMC apoptosis, PPAR-α activators such as


ω-3 fatty acids (e.g., docosahexaenoic acid) may stimulate VSMC apoptosis via a 

p38/PPAR-α-dependent manner. 44 As with ERK1/2, endogenous (retinoic acid) and 

synthetic PPAR ligands (various TZDs such as ciglitazone, troglitazone, and 

rosiglitazone) modulate p38 MAPK activity. 38,45,46 However, both ciglitazone and 

15d-PGJ2 are potent stimulators of p38 MAPK activity in rat astrocytes but not in 

mesangial cells. 45,47 These data demonstrate that PPARs have the ability to directly 

and indirectly regulate MAPKs in a tissue-dependent manner. 

ROLE OF JNK SIGNALING IN VASCULAR REMODELING 

The third member of the MAPK family that we will address is c-Jun N-terminal 

kinase (JNK) which comprises JNK1, JNK2, and JNK3. JNKs can be stimulated 

by several cytokines, growth factors, stressors, and vasoactive agents (Ang II and 

endothelin-1). 26 Contrary to the pro-stimulatory effects of Ang II on cell growth 

via ERK1/2, JNK activation induced apoptosis and cell cycle inhibition. 

Once activated, JNKs activate transcription factors such as c-Jun, c-Myc, 

PPAR-α, and PPAR-γ. 27,48 Ang II-induced JNK activation is dependent upon other 

factors such Ca 2+ mobilization and PKC-ξ activation, but is independent of c-Src 

activation. 49 JNK also seems to be activated in a cell-specific manner. In cardiac 

fibroblasts, Ang II stimulated JNK via PKC-independent proline-rich tyrosine 

kinase 2/Rac1, whereas in VSMCs this occurs through PAK, PKC, and Ca 2+ . 50 

Contrary to the other MAPK proteins, the cellular effects of PPARs on the 

JNK pathway remain to be fully elucidated in VSMCs. PPAR-γ activation may 

induce JNK signaling involved in apoptosis. Taken together, all MAPK pathways 

are involved in molecular mechanisms that ultimately lead to development and 

maintenance of cardiovascular dysfunction such as the vascular remodeling 

observed in hypertension and diabetes. 

PI3K SIGNALING PATHWAY AND VASCULAR REMODELING 

The PI3K pathway is implicated in cardiovascular events such as cardiac and 

vascular remodeling, heart failure, and endothelial dysfunction. 51,52 The PI3K 

family is composed of 3′-OH inositol phosphorylating enzymes and includes 

members of three classes divided according to their molecular structures, activation 

mechanisms, and substrate specificities. Class I (A and B) is composed of 

heterodimeric regulatory and catalytic subunits while class II proteins are composed 

of single catalytic subunits (PI3K-C2α, PI3K-C2β, or PI3K-C2γ). 53 Class 

IA has three catalytic (p110α, β, and δ) and three regulatory subunits (p85α, 

p85β, and p55γ) expressed mostly in the heart and blood vessels. However, class 

IB contains only one protein kinase composed of a p110γ catalytic subunit and 

a p101 regulatory subunit, expressed predominantly in cardiomyocytes, fibroblasts, 

endothelial cells, and VSMCs. 53 

The importance of PI3K in Ang II-induced cardiac hypertrophy and VSMC 

proliferation has been established with the use of specific reversible (LY294002)


and irreversible (wortmannin) inhibitors. 51 PI3K regulates cell growth and survival, 

cytoskeletal reorganization, ROS production, and glucose transport among 

other functions and plays a role in the progression of cardiac hypertrophy and 

dysfunction and in vascular remodeling. 52–54 PI3K is regulated by other kinases 

such as Rho kinase, which may contribute to endothelial dysfunction. 55 PI3K 

participates in endothelial nitric oxide synthase (eNOS) activation and thus in 

NO generation and vasodilation. 56 

Ang II-stimulated ERK1/2 activity was blunted by the PI3K inhibitor LY- 

294009 in mesenteric artery VSMCs from hypertensive rats but not normotensive 

rats, which may imply cross-talk between PI3K and ERK1/2 pathways. 29 PPAR-γ 

regulates PI3K activity. Rosiglitazone induced PI3K/p85α expression and activity 

in adipocytes and in conduit and resistance arteries, but not in skeletal muscle. 

14,57,58 These data suggest that altered PI3K activity and expression may 

occur at the initiation and during the maintenance phase of hypertensive vascular 

disease. 

ROLE OF Akt/PKB SIGNALING PATHWAY IN VASCULAR 

REMODELING 

A key protein within the PI3K signaling pathway is Akt, also known as protein 

kinase B (PKB). In mammals, three Akt/PKB isoforms (Akt1/PKBα, Akt2/PKBβ, 

and Akt3/PKBγ) are highly expressed in cardiac, vascular, and endothelial cells. 53 

They are regulated by PI3K, SH2-containing inositol phosphatase (SHIP2) and 

phosphoinositide-dependent kinase (PDK)-1 and -2. 59 Once activated, Akt/PKB 

modulates cell survival, growth, migration, and glucose metabolism. 60–63 Synthetic 

and endogenous PPAR ligands inhibit Akt/PKB in blood vessels and other 

tissues, possibly through the inhibition of eukaryotic initiation factor-4E-binding 

protein (4E-BP)-1, a protein implicated in cellular growth. 14,64 

ROS AND VASCULAR REMODELING 

Reactive oxygen species (ROS) play important roles in physiological and pathophysiological 

conditions related to cell growth, differentiation, migration and 

signaling, extracellular matrix production and degradation, and inflammation in 

the cardiovascular system. ROS include superoxide anion (•O 2 – ), hydrogen peroxide 

(H 2O 2), hydroxyl radical (•OH – ), and peroxynitrite (ONOO – ) as well as 

other free radicals such as NO. 22,65,66 

Ang II is a critical regulator of ROS generation in many tissues including 

VSMCs and cardiac, mesangial, and endothelial cells. 22 NAD(P)H oxidase is the 

most important vascular source of ROS. It is composed of five subunits: p22 phox , 

gp91 phox (membrane-bound), p47 phox , p67 phox , and a small G protein Rac1 or Rac2 

(cytosolic). In addition to NAD(P)H oxidase, other sources of ROS include 

xanthine oxidase, uncoupled eNOS, and the mitochondrial respiratory chain. 22 

ROS generation in response to pressor doses of Ang II plays a major role in the


deleterious effects of this peptide. These effects are BP-independent effects of 

Ang II since similar BP elevation achieved by norepinephrine infusion did not 

increase vascular •O 2 – production. 67 

The pro-growth and hypertensive effects of ROS have also been shown to be 

mediated by ONOO – production via NO scavenging. 68 Many inhibitors of ROS 

production have been used to elucidate the detrimental role of ROS in hypertension 

and diabetes: protein nitration, lipid oxidation, and DNA degradation. The 

inhibitors include superoxide dismutase (SOD) mimetics (Tempol), •O 2 – scavengers 

(Tiron), specific inhibitors of NAD(P)H oxidase (apocynin, gp91ds-tat), SOD 

inhibitors (PEG-SOD), xanthine oxidase inhibitors (allopurinol) or mitochondrial 

chain inhibitors (thenotrifluoroacetone, carbonyl cyanide-m-chlorophenylhydrazone 

and rotenone). 69–71 Additionally, ROS interact with second messengers (e.g., 

intracellular Ca 2+ ), several kinases such as MAPK (ERK, p38, and JNK) and 

Akt/PKB, and transcription factors (NFκB and AP-1), all leading to the development 

of vascular inflammation and remodeling. 22,72,73 

Several studies have demonstrated beneficial vascular inhibitory effects of 

PPAR activators on ROS production. PPAR-α activators (docosahexaenoic acid, 

DHA, and fenofibrate) and PPAR-γ activators (rosiglitazone or pioglitazone) 

reduced ROS production in rats infused with Ang II and in endothelin-1-dependent 

hypertensive models such as DOCA-salt hypertensive rats. 16,74 

VASCULAR INFLAMMATION AND REMODELING 

One of the major effects of activation of the signaling pathways described is the 

induction of vascular inflammation found in cardiovascular diseases such as 

hypertension and in diabetes. 75 Of the various systems involved, one of the major 

mechanisms is the pro-inflammatory effect of Ang II mediated via the AT 1 receptor. 

75,76 These effects are mediated by increased expression of adhesion molecules 

[intercellular adhesion molecule (ICAM)-1, platelet endothelial cell adhesion 

molecule (PECAM), vascular cell adhesion molecule-1 (VCAM-1), and selectins] 

and transcription factors (NFκB, AP-1) on monocytes, endothelial cells, and 

VSMCs. 13,77,78 

In addition to increased adhesion molecules, Ang II stimulates monocyte 

chemotactic protein (MCP)-1 synthesis in monocytes/macrophages, endothelial 

cells, and VSMCs, causing accumulation of inflammatory cells and molecules in 

the vasculature that ultimately contributes to the development of endothelial 

dysfunction and vascular remodeling. 79 PPAR activators exert potent anti-inflammatory 

effects. For instance, PPAR-α activation with fenofibrate and gemfibrozil 

inhibited cytokine production (TNFα, interferon-γ, interleukin (IL)-6, IL-2, and 

IL-1β) and adhesion molecule synthesis, whereas eNOS and COX-2 expressions 

were increased in VSMCs. 80–82 

These effects were associated with inhibition of expression and activity of 

transcription factors NFκB and AP-1. 83,84 A PPAR-α activator prevented hypertension 

and vascular remodeling by a reduction in NAD(P)H oxidase activity, 

VCAM-1 and ICAM-1 expression, in Ang II infused rats. 74 PPAR-γ also has


potent anti-inflammatory properties in addition to its insulin-sensitizing effects. 

Among beneficial effects reported are reductions in plasminogen activator inhibitor 

(PAI)-1 in microalbuminuria and in matrix metalloproteinase (MMP)-9 along 

with increased NO generation. 13,47,85 The anti-inflammatory vascular effects of 

PPAR-γ occur through inhibition of NFκB and AP-1, as well as down-regulation 

of NAD(P)H oxidase subunits. 13,86 

DUAL PPAR ACTIVATORS 

Because of the beneficial cardiovascular effects of PPAR-α and PPAR-γ activators 

such as improvement of lipid metabolism, insulin sensitization, glucose metabolism, 

vascular remodeling and inflammation, combined activation of these 

nuclear receptors has been attempted. To date, at least eight dual PPARα/γ 

activators are being evaluated in studies ranging from preclinical to phase III. 

The activators have been shown to decrease circulating triglyceride concentrations 

in humans and transgenic human Apo A-I mice. 87,88 

Dual PPAR-α/γ activator effects on glucose metabolism and insulin sensitization 

are similar to those of PPAR-γ activators administered alone. 87,88 Two dual 

PPAR-α/γ activators, ragaglitazar and muraglitazar, demonstrated beneficial 

effects on vascular complications in hypertension and on metabolic abnormalities 

in diabetes, respectively. 89,90 However, muraglitazar administration in type 2 diabetic 

patients was associated with more death and major adverse cardiovascular 

events including myocardial infarction, stroke, and transient ischemic attacks, 

than use of a PPAR-γ activator. 91 

This result dampened interest in these dual PPAR activators, which may not 

really be superior to agents with single receptor activating capability. Because of 

these reported deleterious effects, we and others have used a different approach 

to stimulate both PPAR-α and PPAR-γ receptors. We administered concomitantly 

sub-therapeutic doses of PPAR-α and PPAR-γ activators that had beneficial effects 

when administered in full doses. At these lower doses, a combination of PPARα 

and PPAR-γ administration had beneficial effects in a rodent model of Ang IIinduced 

hypertension on vascular function, including improvement of endothelial 

function, decreased ROS generation, and vascular inflammation. Seber et al. also 

found that concomitant administration of rosiglitazone and fenofibrate improved 

the atherogenic dyslipidemic profiles of type II diabetic patients with poor metabolic 

control. 92 

CONCLUSION 

Hypertension is highly prevalent and one of the major causes of burden of disease, 

particularly cardiovascular morbidity and mortality. Diabetes is increasing worldwide, 

and often is associated with hypertension; it puts hypertensive subjects at 

the highest cardiovascular risk. Both conditions are associated with vascular


injury that serves as the major mechanism for cardiovascular events that lead to 

myocardial infarction, stroke, amputations, and renal failure. 

We have summarized here some of the pathways that participate in growth, 

inflammation, and oxidative stress that lead to atherosclerosis and vascular remodeling 

in hypertension and diabetes. PPAR-α and PPAR-γ appear to act as countervailing 

influences on one of the major activators of the pathways that trigger 

vascular disease, the RAAS. Preclinical and clinical data suggest that PPAR-α 

and PPAR-γ activators may exert important vascular protective effects. Although 

initial experience with agents endowed with combined PPAR-α and PPAR-γ 

stimulatory effects has been disappointing, mechanistic evidence suggests that 

agents with these properties that are able to affect the deleterious intracellular 

signaling pathways described in this chapter may be developed eventually and 

successfully contribute to reducing the burden of disease generated by hypertension 

and diabetes. 

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13 

CONTENTS 

Role of Uncoupling 

Protein 2 in Pancreatic β 

Cell Function: Secretion 

and Survival 

Jingyu Diao, Catherine B. Chan, and 

Michael B. Wheeler 

Abstract .............................................................................................................211 

Introduction .......................................................................................................211 

Regulatory Factors of UCP2 Expression in Pancreatic β Cells ......................212 

UCP2 Action in Insulin Secretion from Pancreatic β Cells: 

Role of ATP .............................................................................................216 

UCP2 Action in β Cell Mass and Survival: 

Role of ATP and ROS Production ..........................................................218 

Conclusion.........................................................................................................219 


References .........................................................................................................220 

ABSTRACT 

Uncoupling protein (UCP) 2 has been considered a negative modulator of insulin 

secretion. In response to stress stimuli such as hyperlipidemia and inflammation, 

the pancreatic β cell up-regulates UCP2 expression, which results in decreased 

insulin secretion. Fatty acids and superoxide regulate UCP2 activity. In addition 

to influencing insulin secretion, UCP2 may play a role in β cell survival and 

proliferation. 

INTRODUCTION 

Regulation of cellular adenosine triphosphate (ATP) production and rapid adjustments 

in ATP levels are essential for most cells during exposure to growth stimuli 

211


or stressful conditions such as fuel deficiency, oxidative stimuli, and inflammation. 

ATP is created by ATP synthase in mitochondria through coupling of the 

proton motive force (PMF) or electrochemical H + gradient to the oxidative phosphorylation 

of fuel substrates such as fatty acids or glucose. To guarantee continuous 

generation of ATP as the main source of energy for cellular function and 

viability, mitochondria operate an oxidative phosphorylation system to supply a 

proton gradient. In this system, PMF is generated during respiration by the 

passage of protons against their gradient through the electron transport chain 

enzyme complexes. Thus, many factors contribute directly to the regulation of 

PMF and thereby ATP synthesis, particularly the carrier proteins in the respiratory 

chain complexes in the inner membranes of mitochondria. 

To maintain an appropriate level of ATP production, an uncoupling process 

that dissipates the proton gradient and prevents the PMF from becoming excessive 

can be recruited. This uncoupling process also plays a role in reducing the level 

of reactive oxygen species (ROS), a by-product of oxidative phosphorylation 

produced by the complexes of the electron transport chain. 1,2 Mitochondrial 

uncoupling is dominated by uncoupling proteins located in the inner mitochondrial 

membranes. 3 Five UCP homologues in mammals sharing distinctive UCP 

signature sequences are thought to be involved in fatty acid anion binding and 

translocation. 1,4 Based on studies of UCP1, the uncoupling activity is augmented 

by fatty acids and inhibited by the purine nucleotide GDP. 5,6 

Specific uncoupling proteins play a well documented, physiological role in 

thermogenesis regulation. UCP1, an uncoupling protein mainly expressed in brown 

adipose tissue, which has little ATP synthase, induces proton leak and reduces ATP 

production, thereby generating heat upon cold exposure and in diet-induced thermogenesis. 

7,8 Activation of UCP1 stimulates respiration, fatty acid oxidation, and 

uncoupling activity. 9 UCP2 and UCP3 share over 50% protein sequence identity 

with UCP1. However, the function and regulation of these UCPs remain unclear. 

UCP3 is expressed in skeletal muscle and brown fat, while UCP2 is found in many 

tissues including heart, brain, kidney, liver, lymphocytes, pancreas, and white adipose 

tissue. 10 The diverse distribution of UCP2 and UCP3 led to the hypothesis 

that their primary physiological function involves something other than the control 

of thermogenesis. 11,12 Specifically, UCP2 appears to play roles in the regulation of 

ROS production, inflammation, and cell proliferation and death. 13,14 

Many recent reviews have discussed the possible physiological and pathophysiological 

roles of UCPs. 15–18 Thus, this chapter focuses on the role of UCP2 

in pancreatic β cell function including the regulation of insulin secretion and cell 

survival. 

REGULATORY FACTORS OF UCP2 EXPRESSION IN 

PANCREATIC β CELLS 

Mitochondrial function is crucial for insulin secretion. 19 For instance, blockade 

of the respiratory chain by mitochondrial toxins inhibits glucose-stimulated

Role of Uncoupling Protein 2 in Pancreatic β-Cell Function 213 

Islet Islet cells 

FIGURE 13.1 UCP2 protein expression in human islets. The islets were fixed with 4% 

paraformaldehyde, permeabilized with 0.3% Triton X-100 detergent and dispersed islet 

cells, and stained with goat antibody specific for UCP2 (Santa Cruz Biotechnology, Inc.). 

Cells were then examined using a Zeiss LSM510 confocal microscope (×40 ×1.3 magnification) 

and images were analyzed using LSM510 image browser software. 

insulin secretion. 20 Absence of specific mitochondrial proteins such as nicotinamide 

nucleotide transhydrogenase (Nnt) in β cells ATP production and insulin 

secretion. 21 Based on the association of the UCP2 gene with obesity and hyperinsulinemia, 

numerous reports have demonstrated the important role of UCP2 in 

the development of type 2 diabetes in humans. 22 

A common –866G/A polymorphism in the promoter of the UCP2 gene 

contributes to diabetes susceptibility in different human populations, perhaps due 

to its overall effects on pancreatic islets, the immune system, and lipid metabolism. 

23,24 Individuals who are heterozygous or homozygous for the –866A allele 

have greater UCP2 activity, which correlates with reduced glucose-induced insulin 

secretion and decreased oxidative stress. 25–27 Immunofluorescence staining of 

UCP2 revealed relatively high amounts of the UCP2 protein in isolated human 

islets, suggesting its importance in islet function (Figure 13.1). However, evidence 

linking any mutations in the UCP2 protein sequence to the pathogenesis of obesity 

or type 2 diabetes 28 is insufficient. 

In contrast, a considerable amount is known regarding the regulation of UCP2 

gene expression. The promoter region of UCP2 gene contains peroxisome


Nutrient Deficiency 

SIRT1 

Reactive 

Oxygen Species 

? 


Inflammatory 

cytokines 

UCP2 

transcriptional/post-transcriptional regulation 

Nutrient Excess 

FIGURE 13.2 Proposed pathways of UCP2 regulation in β cells. UCP2 activity can be 

transcriptionally down-regulated by SIRT1 in response to nutrient deficiency and upregulated 

by signals from ROS, FFAs, and certain pro-inflammatory cytokines due to 

nutrient overload and related insulin resistance. UCP2 can also be activated directly by 

many factors such as ROS and FFA. 

proliferator response elements and a sterol regulatory element. 29–31 In pancreatic 

β cells, UCP2 gene expression is partially controlled by PPAR-γ coactivator PGC- 

1α (Peroxisome Proliferator-Activated Receptor Coactivator 1 Alpha) through 

sterol regulatory element binding protein isoforms (SREBPs). 32,33 Increased activity 

of these transcriptional activators leads to increased UCP2 expression, whereas 

another transcription regulator, SIRT1 (sirtuin1), represses its expression in β 

cells. 34 

SIRT1, a mammalian Sir2 orthologue, is a key regulator implicated in cellular 

stress response and survival through regulation of p53, NF-κB (Nuclear Factor 

KappaB) signaling, and FOXO (Forkhead Box O) transcription factors. 35 A moderate 

reduction in calorie intake (caloric restriction, CR) has been suggested to 

slow aging, reduce age-related chronic diseases, and extend lifespans. In simple 

model organisms, the sir2 gene is a strong candidate to regulate CR. 36 In mammalian 

cells, SIRT1 deacetylase can also be induced by CR, possibly to increase 

the long-term function and survival of critical cell types. 37 

Interestingly, SIRT1 can directly bind to the promoter of UCP2 in response 

to starvation in pancreatic β cells, although it acts on PPAR-γ as well. 34,38 Therefore, 

a decrease in SIRT1 activity in β cells would enhance UCP2 expression and 

reduce insulin secretion, for instance, in response to CR (Figure 13.2). As one 

would expect, the levels of UCP2 are low in sirt1 transgenic mice but high in 

sirt1 knockout mice. 34,39 It is likely, under starvation conditions, that SIRT1 

increases to maintain β cell survival and the cell cycle, leading to the suppression 

of UCP2 expression. Although food deprivation increased UCP2 mRNA in mouse 

pancreas, 34 cells under serum and glucose withdrawal for 15 hr increased SIRT1, 

possibly leading to the suppression of UCP2. 40 Conversely, under obesity or 

FFA 

FFA


Untreated 

TNF IL-1 Palmitate C2-Ceramide 

FIGURE 13.3 Induction of UCP2 protein expression in pancreatic β cell MIN6. Pancreatic 

clonal MIN6 β cells were treated with cytokines (TNF or IL-1, 100 ng/ml), palmitate 

(1 mM), or ceramide (10 μM) for 48 hr, then fixed, permeabilized, and immunofluorescently 

stained with goat anti-UCP2 as indicated in Figure 13.1. 

caloric overload conditions, low levels of SIRT1 result in release of suppression 

on the UCP2 gene, leading to an increase of UCP2 expression. Clearly, more 

research is needed to determine the important link between UCPs and SIRT1 in 

islets. 

Our studies have shown that UCP2 is the predominant isoform expressed in 

murine pancreatic islets, with UCP1 and UCP3 present only at very low levels 

as demonstrated by real-time PCR (unpublished data). Interestingly, in genetically 

obese animal models, animals fed high fat diets, and in clonal MIN6 β cells 

exposed to ROS, free fatty acids (FFAs), or inflammatory cytokines, UCP2 

expression was up-regulated (Figure 13.3), implying that induction of UCP2 in 

β cells directly or indirectly correlates with insulin resistance. 41,42 

This is further supported by our unpublished results showing that the attenuation 

of insulin signaling by chronic silencing of the insulin receptor in pancreatic 

cells increases the expression of UCP2 protein and augments mitochondrial 

uncoupling activity. Together, these data suggest that β cells may adapt to obesity 

and insulin resistance by increasing UCP2 levels and thus uncoupling activity, 

consistent with the notion that UCP2 expression adapts to the metabolic state of 

an organism in a tissue-specific manner. 43 This adaptation with insulin resistance 

would result in the attenuation of insulin secretion from β cells in response to 

increased glucose (Figure 13.2). 

Because UCP2 is up-regulated in β cells in response to insulin resistant states, 

identifying signaling pathways regulating its expression will facilitate understanding 

its function at the molecular level. Along these lines, we have observed that 

interleukin 1(IL-1), a primary regulator of inflammatory and immune responses, 

increases UCP2 protein expression in mouse clonal MIN6 β cells without causing 

significant cytotoxic effects (Figure 13.3). In contrast, in rat clonal INS-1 β cells, 

IL-1β down-regulated UCP2 mRNA accompanied by decreased cell viability,


suggesting that IL-1 can differently regulate UCP2 expression in β cells depending 

on cell signaling status. 44 

IL-1 binds to cell surface-specific receptors, activating specific protein kinases 

such as NIK (NF-κB Induced Kinase) and MAPK (Mitogen-Activated Protein 

Kinase), and modulates transcription factors including NF-κB, AP-1 (Adaptorrelated 

Protein Complex 1) and CREB (cAMP Responsive Binding Protein 1), 

resulting in the expression of immediate early genes central to the inflammatory 

response. 45 Therefore, the fact that UCP2 protein expression can be up-regulated 

by IL-1 suggests that UCP2 is one of the acute response genes in β cells. 

Consistent with this finding, IL-1β is a key inflammatory mediator causing pancreatic 

islet dysfunction and apoptosis through the up-regulation of inducible 

nitric oxide synthase and cyclooxygenase-2. 46 Although IL-1 is produced mainly 

by monocytes, macrophages, and other cell types, human β cells make IL-1β in 

response to high glucose concentrations independently of an immune-mediated 

process. However, the β cells also express IL-1 receptor antagonist (IL-1Ra), a 

naturally occurring anti-inflammatory cytokine, to protect themselves from glucotoxicity. 

47,48 The pathway of IL-1-mediated β cell UCP2 regulation is therefore 

a potential target for controlling β cell function and survival. 

UCP2 ACTION IN INSULIN SECRETION FROM 

PANCREATIC β CELLS: ROLE OF ATP 

The β cell is essential for glucose homeostasis due to its ability to secrete 

insulin in response to nutrients. 49 In general, glucose metabolism in ß cells 

leads to an elevated intracellular ATP:ADP ratio, which closes the ATP-sensitive 

K + (K ATP) channel, resulting in depolarization of the cell membranes. Closure 

of K ATP channels activates voltage-dependent Ca 2+ channels, leading to Ca 2+ 

entry and insulin exocytosis. 50 Thus, given the key role ATP plays in mediating 

insulin secretion and synthesis, it is not surprising that UCP2 negatively regulates 

insulin secretion since its uncoupling activity reduces mitochondrial ATP 

production. 

Our laboratory and others have shown that cellular ATP levels in UCP2 –/– 

animals are elevated in association with enhanced insulin secretion, 42,51 while 

decreased ATP levels and impaired glucose-stimulated insulin secretion occur 

when UCP2 is overexpressed. 42,52,53 Furthermore, it appears that increased islet 

UCP2 levels occur in several rodent models of type 2 diabetes and β cell dysfunction, 

including hyperglycemic HFD-fed mice, ob/ob mice, fa/fa rats, mice 

overexpressing SREBP-1c in β cells, and rodents expressing a dominant-negative 

IGF-1 receptor in skeletal muscle. 42,53a–57 

In addition to transcriptional regulation of UCP2 expression, regulatory mechanisms 

that occur more rapidly are important because they allow cells to rapidly 

adjust ATP levels in response to various stimuli or stresses. Post-transcriptional 

mechanisms regulate UCP2 activity, suggesting that UCP2 is able to respond 

acutely to stimuli. 58 Furthermore FFAs and superoxide radicals directly activate


Mitochondrial 

membrane potential 

(Safranin red (RFU)) 

40000 

30000 

20000 

10000 

Gl-3-P 7.5mM 

OA 25µM 

OA 75µM total 

OA 50µM total 

FCCP 5.6µM 

0 100 200 300 400 500 

600 

Time (sec) 

Control 

UCP2 

UCP1 

Depol. and 

Uncoupling 

Hyperpol. 

FIGURE 13.4 FFA activates mitochondrial uncoupling. Pancreatic clonal MIN6 β cells 

were infected with recombinant adenovirus expressing UCP1, UCP2, and control green 

fluorescence protein for 48 hr, then permeabilized and subjected to measurement of 

mitochondrial membrane potential in a buffer containing 2.5 μM safranin. Mitochondrial 

membrane potential as reflected by the fluorescence was monitored by a FluoroCount plate 

reader at excitation/emission wavelengths of 530/590 nm. Respiratory substrate glycerol- 

3-phosphate (Gl-3-P, 7.5 μM) was first added to induce mitochondrial hyperpolarization. 

To induce OA-mediated uncoupling effects, free fatty acid oleate (OA) was added to build 

up OA concentration from 25 to 75 μM. To confirm the depolarization of mitochondrial 

membrane, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was added to a 

final concentration of 5.6 μM. Vertical arrows denote time points of chemical additions. 

(Courtesy of Vasilij Koshkin, Wheeler Laboratory.) 

uncoupling. Mitochondrial superoxide activates UCP2-mediated proton leaks and 

influences ATP production and insulin secretion in β cells. 59–64 

Using safranin fluorescence to measure mitochondrial membrane potential, 

we demonstrated that 50 to 75 μM oleate acutely induced UCP2-mediated uncoupling 

within seconds in MIN6 cells, suggesting that UCP2 activity can be induced 

by FFAs directly or by an immediate consequence of ROS generation and lipid 

peroxidation of FFAs (Figure 13.4). Although the mechanism of FFA-induced 

UCP2 activation remains unclear, it presumably occurs through direct binding of 

FFA to UCP2. 1 

The links of UCP activators and their acute uncoupling effects on mitochondria 

are interesting and suggest an avenue for future investigations that may shed 

light on the function of UCP2. It should be noted that acute exposure of pancreatic 

cells to both high glucose concentrations and saturated FFAs stimulates appreciable 

insulin secretion, whereas chronic exposure results in desensitization and 

suppression of secretion. 65 Binding of FFA to its receptor GPR40 would increase 

intracellular cAMP levels and stimulate Ca 2+ influx through voltage-dependent 

Ca 2+ channels, resulting in an increase in insulin secretion. 66


The effect from the FFA–GPR40 pathway may override the effect from 

FFA–UCP2 activity initially, considering that uncoupling has a negative impact 

on FFA-stimulated insulin secretion. However, it is also possible that UCP2 may 

initially promote insulin secretion but may ultimately attenuate it. Which of these 

opposing effects predominates? This is difficult to determine since UCP2 protein 

levels do not necessarily reflect levels of activity. Specifically, UCP2 appears to 

require activation by fatty acids, and studies demonstrating a quantitative correlation 

between UCP2 activity and insulin secretion in vivo have yet to be performed. 

Nevertheless, it is conceivable that in metabolic states where fuel is 

plentiful, increased UCP2 expression and activity by factors such as pro-inflammatory 

cytokines and high circulating FFA levels are highly correlated to the 

desensitization of β cells to release insulin. 

UCP2 ACTION IN ββ CELL MASS AND SURVIVAL: 

ROLE OF ATP AND ROS PRODUCTION 

In type 2 diabetes, reduced β cell mass is a key feature associated with defects 

in insulin secretion. 61 The role of UCP2 in ATP production and ROS regulation 

has linked this protein not only to β cell function, but also to survival and 

proliferation. Increased ROS are thought to induce insulin resistance in numerous 

settings. 68 Undoubtedly, excessive ROS production is harmful to cell viability. 

However, moderately elevated ROS levels can also be essential for activating 

defensive signaling pathways to protect cells. 69 

For example, ROS can protect against ischemia–reperfusion injury in cardiomyocytes. 

2,69 The physiological role behind this phenomenon is that mitochondrial 

ROS is triggered to promote signaling pathways for gene transcription and 

cell growth in cells with low energy states. ROS may function as mediators to 

facilitate efficient energy transfer between mitochondria and ATPases in cells 

with high energy states. 70 Therefore, the beneficial effect of having lower levels 

of uncoupling activity in certain cell types like β cells under normal physiologic 

conditions may be to maintain a reasonable amount of ROS production. However, 

under pathological conditions when ROS production becomes excessive and 

harmful to cells, UCP2 may induce uncoupling of mitochondria in order to reduce 

ROS production. 

In support of this, evidence suggests that UCP2 controls mitochondrial ROS 

production which, in turn, confers neuroprotection, cardioprotection, and life span 

extension in Drosophila. 14,71,74 In the pancreatic β cell line INS-1, overexpression 

of UCP2 improved cell survival when the cells were treated with H 2O 2. 75 However, 

under chronic metabolic stress, even modestly increased levels of UCP2 protein 

may promote cell death in other types of tissues and cells including liver cells 

and Hela cells. 76–78 

With respect to cell proliferation, based on our study, UCP2 overexpression 

in MIN6 cells not only reduces mitochondrial ROS formation, but also increases 

proliferation under both starvation and nutritional excess conditions (unpublished


data). Consistent with these observations, Ucp2 –/– mice had increased islet masses 

after 4.5 months of high fat diet feeding, suggesting that UCP2 may play a role 

in the regulation of β cell growth, probably by directly augmenting FFA-mediated 

ATP production. 51 The potential effect on cell growth is supported by the tumor 

formation that occurs upon deletion of UCP2 in association with increased NFκB 

action and oxidative stress. 79 

Islets of UCP2 –/– mice showed increased ROS levels when compared with 

islets from wild type (WT) mice. 80 In addition, FFAs also increased ROS production 

in isolated islets from WT and cultured pancreatic β cells, but not in 

islets from UCP2 –/– mice, 80,81 suggesting UCP2 contributes to the regulation of 

ROS production in β cells. However, data from our laboratory also suggest that 

despite the role of UCP2 in β cells, other factors may be involved in β cell 

regulation of intracellular ROS. Specifically, induction of ROS formation in vitro 

using menadione, ceramide, or cytokines in both Ucp2 –/– and WT islet cells failed 

to reveal any protective role of UCP2, perhaps because these mediators act beyond 

the UCP2 pathway (unpublished data). In addition, Ucp2 –/– and WT islets had 

equal sensitivities to apoptosis induced by menadione, ceramide, and cytokines; 

overexpression of UCP2 did not protect MIN6 cells from either cytokine- or 

menadione-mediated apoptosis. Thus, these data suggest that pancreatic β cells 

may have a specific regulatory signaling network to maintain their own levels of 

ROS, which may only be partially regulated by UCP2. 

CONCLUSION 

Through its fundamental role in the regulation of mitochondria ATP generation 

and ROS production, UCP2 is a key regulator of β cell function and survival. 

The pharmacological targeting of UCP2 in β cells presents a potential way to 

modulate insulin secretion and β cell proliferation. However, β cell-specific 

signaling pathways mediating the induction, activation, and effects of UCP2 need 

to be identified to enable precise manipulation without deleterious side effects. 


This work was funded by Grants MOP 12898 and MOP 43987 from the 

Canadian Institutes of Health Research (CIHR) to M.B.W. and C.B.C, respectively. 

M.B.W. is supported by an investigator award from CIHR. J.D. was 

supported by a fellowship award from the Canadian Diabetes Association. J.W. 

Joseph, V. Koshkin, A.V. Gyulkhandanyan, S.C. Lee, G.T. Karaman, C. Thorn, 

and G. Bikopoulos are gratefully acknowledged as contributors to the work 

presented here. We thank D. Yau, E. Allister, and N. Wijesekara for help with 

editing of the manuscript.


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Section II 

Influence of Dietary Factors, 

Micronutrients, and 

Metabolism

14 

CONTENTS 

Nutritional Modulation 

of Inflammation in 


Uma Singh, Sridevi Devaraj, and Ishwarlal Jialal 

Abstract .............................................................................................................227 

Metabolic Syndrome.........................................................................................228 

Metabolic Syndrome and Cardiovascular Disease...........................................228 

Metabolic Syndrome and Diabetes...................................................................229 

Inflammation, Metabolic Syndrome, and Acute Phase Proteins......................229 

Pro-Inflammatory Cytokines, Monocytes, and Metabolic Syndrome..............230 

Adipose Tissue and Inflammation ....................................................................231 

Therapeutic Modulation of Inflammation in Metabolic Syndrome.................232 

Weight Loss, Hypocaloric Diets, Inflammation, and Metabolic Syndrome....232 

Pharmacological Therapies with Potential to Prevent or Treat 

Metabolic Syndrome ...............................................................................237 

Conclusion.........................................................................................................238 

References .........................................................................................................238 

ABSTRACT 

Metabolic syndrome (MetS) is a disorder composed of central adiposity, dyslipidemia, 

abnormal glucose tolerance, and hypertension. It confers an increased 

risk for diabetes and cardiovascular disease (CVD). Inflammation plays a pivotal 

role in atherosclerosis and is involved in abnormalities associated with MetS such 

as insulin resistance (IR) and adiposity. The various biomarkers of inflammation 

such as inflammatory cytokines (TNF-α, IL-6, and IL-1β), chemokines (MCP-1 

and IL-8), and C-reactive protein (CRP) are increased in obesity and correlate 

with IR and CVD. IR is also associated with endothelial dysfunction (ED). 

Inflammation, IR, and ED amplify the cascade of metabolic and vascular 

derangements. The etiology of this syndrome is largely unknown but presumably 

represents a complex interaction of genetic and environmental factors. Since MetS 

is associated with chronic low grade inflammation, strategies are being explored 

227


to ameliorate its pro-inflammatory status. MetS has been identified also as a target 

for diet therapy to reduce risk of CVD. Weight loss appears to be the best modality 

to reduce inflammation. Intervention trials convincingly demonstrate that weight 

loss and/or increased physical activity reduce biomarkers of inflammation such 

as CRP and IL-6. Thus, therapeutic lifestyle change (TLC) is strongly suggested 

for MetS subjects both for weight reduction and also to reduce the inflammation 

associated with this syndrome. Much more research is needed to define the roles 

of individual dietary factors on the biomarkers of inflammation and the mechanisms 

of the anti-inflammatory effects of weight loss in MetS. 


MetS comprises a cluster of abnormalities with insulin resistance and adiposity 

as its central features. 1–3 Five diagnostic criteria were identified by the Adult 

Treatment Panel III in the executive summary of a report of the National Cholesterol 

Education Program (NCEP). The presence of any three of the five features 

is considered sufficient to diagnose MetS. 4 These include central obesity, high 

triglycerides, low HDL, hypertension, and impaired fasting glucose. Using the 

NCEP definition on a representative sample of 8814 men and women from the 

United States, the age-adjusted prevalence of MetS was 24% in men and 23.4% 

in women. 5 Applying this figure to the U.S. population in the year 2000 means 

about 47 million residents have MetS. 

Recently, a worldwide consensus for the MetS defined by the International 

Diabetes Federation (IDF) includes central obesity as a main component and the 

other factors such as hypertension, dyslipidemia, and impaired fasting glucose 

as other components, all of which lead to insulin resistance and independently 

predict cardiovascular events. 6 The National Heart, Lung, and Blood Institute/American 

Heart Association statement defining MetS has just been published. 

It encompasses the recent IDF guidelines, but includes central obesity, dyslipidemia, 

hypertension, and impaired fasting glucose as the features. 7 This chapter 

will focus on biomarkers of inflammation in relation to MetS and its nutritional 

modulation. Particular emphasis will be given to nutritional insights derived 

from therapeutic interventions with diet and exercise in subjects with metabolic 

abnormalities. 

METABOLIC SYNDROME AND CARDIOVASCULAR DISEASE 

Subjects with MetS have increased burdens of cardiovascular disease. In the 

Kuopio Ischemic Heart Disease Study, Lakka et al. 8 convincingly showed that 

men with MetS, even in the absence of baseline CAD or diabetes, had significantly 

increased mortality from CAD. In the Botnia Study, 9 MetS was defined as the 

presence of at least two of the following risk factors: obesity, hypertension, 

dyslipidemia, and microalbuminuria. Cardiovascular mortality was assessed in 

3606 subjects with a median follow-up of 6.9 years. In women and men,

Nutritional Modulation of Inflammation in Metabolic Syndrome 229 

respectively, MetS was seen in 10 and 15% of subjects with normal glucose 

tolerance (NGT), 42 and 64% of those with impaired fasting glucose/impaired 

glucose tolerance (IFG/IGT), and 78 and 84% of those with type 2 diabetes 

mellitus. The risk for coronary heart disease and stroke was increased three-fold 

in subjects with MetS (p


markers and several features of MetS by conducting Z-score analyses in 107 nondiabetic 

subjects. CRP levels were shown to be strongly associated with insulin 

resistance calculated from the HOMA model, blood pressure, low HDL, triglycerides, 

and levels of the IL-6 and TNF pro-inflammatory cytokines. Body mass 

index (BMI) and insulin resistance were the strongest determinants of the inflammatory 

state. 

Festa et al. 15 who participated in the Insulin Resistance and Atherosclerosis 

Study (IRAS) showed that hsCRP was positively correlated with BMI, waist 

circumference, and other individual risk factors of MetS. Multivariate linear 

regression models reported the strongest correlation of CRP with BMI, systolic 

blood pressure, and insulin sensitivity index. All these studies record the universal 

observation that CRP is elevated in subjects with features of MetS and indicate 

a linear relationship between CRP levels and number of MetS features. Furthermore, 

the strongest associations are observed between CRP levels and central 

adiposity and insulin resistance. 

PRO-INFLAMMATORY CYTOKINES, MONOCYTES, AND 


Cytokines, in particular IL-1, TNF, and IL-6, are the main inducers of the acute 

phase response (APR). 21 Several lines of evidence indicate that IL-6 is the central 

mediator of the inflammatory response and promotes insulin resistance. 22 In 

addition, IL-6 administered to humans subcutaneously induces an acute inflammatory 

response. 23 In fact, IL-6 is believed to be the main driver of CRP release 

from hepatocytes. 24 

A very tight correlation exists between IL-6 and CRP; such a strong correlation 

does not exist for other cytokines. Furthermore, IL-6 and hsCRP are 

associated with visceral adiposity and 30% of circulating IL-6 is derived from 

human adipose tissue. 25 Moreover, baseline IL-6 levels independently predict 

future CVD. 26 Pickup et al. 19 demonstrated increased levels of IL-6 in subjects 

with more than two features of MetS. Our group also showed that monocyte 

release of IL-6 in type 2 diabetes is significantly increased when compared to 

non-diabetic controls. 16 Several studies have shown that TNF secreted by monocytes–macrophages, 

endothelial cells, and also to a large extent by adipose tissue, 

is an important regulator of insulin sensitivity. 27 

Elevated IL-18 level has been recently reported as an independent risk predictor 

for MetS in the absence of a history of type 2 diabetes in a large communitybased 

population sample. 28 IL-18 functions as a pleiotropic pro-inflammatory 

cytokine and stimulates the production of TNF and secondarily IL-6. IL-18 may 

form a link between MetS and atherosclerosis because it is highly expressed in 

atherosclerotic plaques, and its role in plaque destabilization has been suggested. 29 

Additionally, data from the recently reported MONICA/KORA study show that 

elevated levels of IL-18 are associated with considerably increased risks of type 

2 diabetes. 30


Chemokines also play an important role in leukocyte trafficking. Circulating 

levels of chemokines have been shown to increase inflammatory processes including 

obesity-related pathologies. Kim et al. 31 reported that circulating levels of 

MCP-1 and IL-8 are related to obesity-related parameters such as BMI, waist 

circumference, CRP, IL-6, HOMA, and low HDL-cholesterol. These findings 

suggest that the circulating MCP-1 and/or IL-8 may be a potential candidate 

linking obesity with obesity-related metabolic complications such as atherosclerosis 

and diabetes. 

Since low grade inflammation is a feature of MetS, it is important to state 

that the pro-inflammatory status appears to result from an imbalance between 

pro- and anti-inflammatory cytokines. Importantly, in the Leiden 85-Plus Study, 

subjects who developed diabetes and had more than three MetS features had 

lower levels of LPS-stimulated whole blood release of IL-10 (a potent antiinflammatory 

cytokine) than those who did not. 32 Esposito et al. 33 also reported 

that in both obese and non-obese women, IL-10 levels were significantly lower 

in women with MetS. 

ADIPOSE TISSUE AND INFLAMMATION 

Changes in adipose tissue (AT) mass are associated with altered endocrine and 

metabolic functions of the adipose tissue. 34 Most importantly, adipose tissue is a 

depot of inflammatory molecules, collectively called the adipocytokines or adipokines, 

including tumor necrosis factor (TNF), angiotensinogen, PAI-1, leptin, 

and adiponectin. Unlike adipose tissues of lean individuals, adipose tissues of 

obese subjects secreted increased amounts of TNF, IL-6, iNOS, TGF-β, CRP, 

sICAM, MCP-1, procoagulant proteins such as PAI-1, and tissue factor. 35 Elevated 

PAI-1 levels, the principal inhibitors of fibrinolysis, have been reported in many 

clinical and population studies in obese subjects and correlate with abdominal 

patterns of obesity 36 and other components of MetS. Furthermore, it appears to 

be even a better predictor of the MetS status than CRP. 

A large body of evidence documents low levels of adiponectin in subjects 

with MetS. 37 Adiponectin belongs to the soluble defense collagen family and has 

been shown to suppress expression of adhesion molecules by endothelial cells, 

lipid accumulation, TNF release from monocytes, and vascular smooth muscle 

cell proliferation; it also reduces atherosclerotic vascular lesions in vivo. Adiponectin 

is an atypical adipokine because in contrast to the dramatic increase in 

plasma levels of all other adipokines, the circulating concentration of adiponectin 

is paradoxically decreased in obesity. 

A strong correlation between low levels of adiponectin and increased insulin 

resistance is well established in humans. 38 Adiponectin also improves insulin 

resistance in vivo. Among Asian Indians, plasma levels of adiponectin in subjects 

with IGT are strong predictors of the development of diabetes. 39 In a step-wise 

regression model of a study examining the association between CRP and adiponectin 

levels, hsCRP was independently associated inversely with levels of


TABLE 14.1 

Metabolic Syndrome: Evidence for a 

Pro-Inflammatory State 

↑ Acute phase proteins (HsCRP and SAA) 

↓ Adiponectin 

↑ Pro-inflammatory status (IL-6, IL-18, and TNF-α) 

↑ Leptin 

↑ Chemokines (MCP-1 and IL-8) 

↓ Anti-inflammatory cytokines (IL-10) 

adiponectin and positively with levels of leptin. 40 Adiponectin exerts anti-atherogenic 

properties by suppressing the endothelial inflammatory response, inhibiting 

TNF and monocyte adhesion, and suppresses transformation of macrophages to 

foam cells. 

THERAPEUTIC MODULATION OF INFLAMMATION IN 


Inflammation can be reduced by a variety of approaches including diet, exercise, 

and pharmacotherapy (statins, PPAR-α and -γ agonists, and the endocannabinoid 

receptor-1 blocker, rimonabant). As reviewed previously 41 and summarized in 

Table 14.1, people with MetS typically manifest pro-thrombotic and pro-inflammatory 

states. Therapeutic lifestyle changes (TLCs), including diet and exercise, 

serve as cornerstone therapies for the treatment of MetS. Thus, the first step in 

reducing the excess cardiovascular risk associated with MetS is the adoption of 

a healthier lifestyle (particularly reducing body weight and increasing physical 

activity). 

WEIGHT LOSS, HYPOCALORIC DIETS, INFLAMMATION, AND 


Weight losses ranging from 3 to 15 kg achieved through different diet programs 

(low fat, high protein, or hypocaloric diets) result in concomitant reductions of 

CRP levels by 7 to 48% as shown in several studies. We recently reviewed the 

literature 42,43 and documented a significant positive correlation (r 2 = 0.8693; p 


changes on markers of systemic vascular inflammation and IR, they conducted 

a randomized single-blind trial in 120 premenopausal (aged 20 to 46 years) obese 

women (BMI ≥30 kg/m 2 ) without diabetes, hypertension, or hyperlipidemia. The 

intervention group (n = 60) adhered to a low energy Mediterranean-style diet 

(foods rich in complex carbohydrates, monounsaturated fat, and fiber; lower ratios 

of omega-6 to omega-3 fatty acids) and increased physical activity. The control 

group (n = 60) was given general information about healthy food choices and 

exercise. BMI decreased significantly more in the intervention group than in 

controls, as did serum concentrations of IL-6, IL-18, and CRP, while adiponectin 

levels increased significantly. In multivariate analyses, changes in FFA and adiponectin 

levels were independently associated with changes in insulin sensitivity 

The same group of authors 45 investigated the effects of Mediterranean-style 

diets on endothelial function and vascular inflammatory markers in 180 patients 

with MetS. The 99 men and 81 women with MetS, as defined by the ATP III, 

were randomized equally in the intervention and placebo groups for 2 years. The 

patients in the intervention group were instructed to follow a Mediterranean-style 

diet and received detailed advice about how to increase daily consumption of 

whole grains, fruits, vegetables, nuts, and olive oil. Patients in the control group 

followed a prudent diet (50 to 60% carbohydrates, 15 to 20% proteins, total fat 


TABLE 14.2 

Randomized Clinical Trials Examining Effects of Lifestyle Interventions 

(Diet and Exercise) on Biomarkers of Inflammation 

Study 

Investigators, 

Duration, 

Reference Subjects 

Esposito et al. 

2003 

(2 years) 44 

Esposito et al. 

2004 

(2 years) 45 

Tchernof et al. 

2002 

(14 months) 46 

Orchard et al. 

2005 

(3.2 years) 48 

Seshadari et al. 

2004 

(6 months) 50 

Kopp et al 

2003 

(14 months 

after surgery) 51 

120 premenopausal 

obese women (20 to 

46 years, BMI ≥30 

kg/m 2 ) 

180 MetS patients (99 

men and 81 women) 

61 obese, 

postmenopausal 

women (mean age 

56.4 years, BMI-35.6 

kg/m 2 ) 

Participants had IGT; 

657 controls and 638 

subjected to 

intervention 

78 severely obese 

patients, 86% with 

diabetes and/or MetS 

37 morbidly obese 

patients (BMI = 49 

kg/m 2 ) 

Lifestyle Intervention 

Type 

Mediterranean-style diet 

(rich in complex 

carbohydrates, MUSFAs 

and fiber, lower ratio of 

omega 6/omega 3) and 

increased physical activity 

versus control subjects 

informed of healthy food 

choices and exercise 

Mediterranean style diet 

versus prudent diet (50 to 

60% carbohydrates, 15 to 

20% proteins,


TABLE 14.2 (CONTINUED) 

Randomized Clinical Trials Examining Effects of Lifestyle Interventions 

(Diet and Exercise) on Biomarkers of Inflammation 

Study 

Investigators, 

Duration, 

Reference Subjects 

Troseid et al. 

2004 

(12 weeks) 54 

Troseid et al. 

2005 

(12 weeks) 55 

Roberts et al. 

2005 

(3 weeks) 56 

Brinkworth et 

al. 2004 

(12 weeks 

energy 

restriction + 4 

weeks energy 

balance + 52 

weeks with 

minimal 

professional 

support) 57 

15 MetS subjects in 

exercise (n = 9) and 

non-exercise (n = 6) 

groups 

Lifestyle Intervention 

Type 

Endurance exercise 

(walking or jogging on 

treadmill) and strength 

training 45 to 60 minutes 

three times weekly in 

training studio under 

supervision 

32 subjects with MetS 2 × 2 randomized factorial 

trial, physical exercise 

31 obese patients; 15 

with MetS 

58 (13 male, 45 female) 

obese non-diabetic 

subjects with 

hyperinsulinemia; 

mean age =50 years, 

BMI = 34 kg/m 2 

IGT = impaired glucose tolerance. APN = adiponectin. 

High fiber, low fat diet; 3week 

residential program 

(ad libitum food and daily 

aerobic exercise) 

Randomization to standard 

protein (15% protein, 55% 

carbohydrate) or high 

protein (30% protein, 40% 

carbohydrate) diet 

Changes in Biomarkers 

of Inflammation 

Significant reduction in 

MCP-1 and IL-8 

No change in CAMs 

Significant reductions in 

CRP, sICAM, sP-selectin, 

MIP-1, MMP-9; 

increased NO; significant 

reduction in monocyte 

adhesion and chemotactic 

activity 

Significant (


reviewed the importance of moderate increases in physical activity along with a 

detailed and tailored Mediterranean-style diet to reduce the prevalence of MetS 

and associated cardiovascular risks through reducing systemic inflammation and 

endothelium dysfunction, particularly in patients who failed to lose weight. 

In obese women, hypocaloric diets have been associated with weight reduction 

as well as inflammation. In this context, Seshadari et al. 50 found overall 

favorable effects on inflammation in 78 severely obese subjects from a low 

carbohydrate diet (LCD) versus a conventional (fat- and calorie-restricted) diet 

for a period of 6 months; 86% of the subjects were either diabetic or had features 

of MetS. Overall, CRP levels decreased modestly in both diet groups. However, 

patients with high risk baseline levels (CRP >3 mg/dL, n = 48) experienced 

greater decreases in CRP on a low carbohydrate diet, independent of weight loss. 

Kopp et al. 51 examined the cross-sectional and longitudinal relationships of 

CRP, IL-6, and TNF with features of the IRS in 37 morbidly obese patients (BMI 

= 49 kg/m 2 ) with different stages of glucose tolerance before and 14 months after 

gastric surgery. Weight loss after gastric surgery induced a significant shift from 

diabetes (37 versus 3%) to IGT (40 versus 33%) and NGT (23 versus 64%). 

Concentrations of CRP and IL-6 decreased after weight loss, whereas serum 

levels of TNF-alpha remained unchanged. It was concluded that weight loss 

results in a marked decrease in circulating levels of inflammatory markers in 

association with a reversal of diabetes in morbidly obese individuals after gastroplastic 

surgery. However, long-term studies are needed to show whether this 

improvement in cardiovascular risk factors will eventually translate into a significant 

clinical benefit with regard to cardiovascular morbidity and mortality. 

Cellular adhesion molecules (CAMs) such as E-selectin, intercellular adhesion 

molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) 

are involved in the rolling, adhesion, and extravasation of monocytes and T 

lymphocytes into the atherosclerotic plaque. Serum concentrations of CAMs are 

higher in patients with coronary artery disease than in healthy control subjects. 52 

Moreover, male participants in the Physician’s Health Study who had ICAM-1 

levels in the highest quartile are at greater cardiovascular risk than men in the 

lowest quartile. 53 In line with these findings, Troseid et al. 54 published a study 

conducted as an unmasked randomized 2 × 2 factorial trial involving an intensive 

exercise protocol of 12 weeks’ duration. In the combined exercise groups, significant 

reductions in MCP-1 and IL-8 as compared to the combined non-exercise 

groups were noted along with a significant reduction versus baseline for both 

chemokines. The changes in MCP-1 were significantly correlated to changes in 

visceral fat. 

However, the same group of investigators 55 recently reported a negative study 

with regard to the effect of physical exercise on serum levels of CAMs. These 

authors explored the possible role of adipose tissue in regulating serum levels of 

CAMs. No significant changes in CAMs were observed in the intervention group. 

On examination of the whole study population regardless of intervention, changes 

in serum E-selectin were significantly correlated to changes in body mass index, 

waist circumference, fasting glucose, and HbA1c, but not to changes in visceral


fat, subcutaneous fat, TNF, or adiponectin. Thus this study highlights changes in 

glycemic control and obesity rather than regional fat distribution to influence Eselectin 

levels in subjects with MetS. 

Additionally, Roberts et al. 56 recently reported the results of examining the 

effects of lifestyle modification on various key contributing factors to atherogenesis, 

including oxidative stress, inflammation, chemotaxis, and cell adhesion. 

Obese men (n = 31), 15 of whom had MetS, were placed on a high fiber, low fat 

diet in a 3-week residential program where food was provided ad libitum and 

daily aerobic exercise (45 to 60 min) was performed. After 3 weeks, significant 

reductions in BMI, CRP, sICAM-1, sP-selectin, MIP-1α, MMP-9, and biomarkers 

of oxidative stress with concomitant increases in NO production were noted. 

Additionally, both monocyte adhesion and monocyte chemotactic activity (MCA) 

significantly decreased. Nine of 15 subjects were no longer positive for MetS 

post-intervention. This study postulated the amelioration of CAD risk factor by 

intensive lifestyle modification in men with MetS factors prior to reversal of 

obesity. 

Comparative effects of two low fat diets differing in carbohydrate-to-protein 

ratio for biomarkers of CVD risk in obese subjects with hyperinsulinemia were 

examined. 57 Two groups totaling 58 obese, non-diabetic subjects with hyperinsulinemia 

(13 males and 45 females, mean age 50.2 years, mean BMIs of 34.0 

kg/m 2 , mean fasting insulin levels of 17.8 mU/l) were randomly assigned to either 

a standard protein (SP; 15% protein, 55% carbohydrate) or high protein (HP; 

30% protein, 40% carbohydrate) diet during 12 weeks of energy restriction 

(approximately 6.5 MJ/day) and 4 weeks of energy balance (approximately 8.3 

MJ/day). They were subsequently asked to maintain the same dietary pattern for 

the succeeding 52 weeks with minimal professional support. The measurements 

included fasting blood lipids, glucose, insulin, and CRP and sICAM-1 at baseline 

and at weeks 16 and 68. In total, 43 subjects completed the study with similar 

dropouts in each group. At week 68, there was net weight loss (SP –2.9 ± 3.6%; 

HP –4.1 ± 5.8%; p


Chief among these are statins, insulin sensitizers such as metformin and 

thiazolidinediones (rosiglitazone and pioglitazone), fibrates, and a newly discovered 

drug named rimonabant. In various large prospective studies, statins have 

been shown to reduce hsCRP. 59 Rosiglitazone has also been shown to significantly 

reduce hsCRP levels, 60 suggesting that PPAR-γ agonists may reduce markers for 

subclinical inflammation to the levels seen in statin trials. Furthermore, therapy 

with fenofibrate, a fibric acid derivative, has been shown to lower IL-6 and hsCRP 

in borderline hyperlipidemic subjects. 61 In addition, the endocannabinoid system 

appears to play a key role in metabolism and weight gain. The investigational 

rimonabant is a cannabinoid receptor type 1 blocker that has been employed in 

trials involving more than 6500 patients. It has resulted in reduction in the 

prevalence of MetS as well as biomarkers of inflammation along with improved 

glycemic and lipid profiles. 58,62 

CONCLUSION 

It appears from the available literature that therapeutic lifestyle change remains 

the cornerstone in modulating inflammation and CVD. If lifestyle change is not 

sufficient, then drug therapies for abnormalities in individual risk factors are to 

be indicated. To date, we have insufficient evidence for primary use of drugs that 

target the underlying causes of MetS. Weight loss and hypocaloric diets are 

definitely associated with reduced inflammation and overall risk reduction of 

CVD. 

Also, most studies reported effects on CRP, whereas a few focused on proinflammatory 

cytokines such as IL-6, IL-18 or TNF-α and chemokines (IL-8 and 

MCP-1). Future research should focus on the roles of specific nutritional components 

in various diets on biomarkers of inflammation, as they may modulate 

inflammation through different mechanisms. Despite the fact that precise mechanisms 

have yet to be established, both diet and physical activity play pivotal 

roles in improving many factors associated with MetS including modulation of 

various adipocytokines. A more thorough understanding of the clustering of 

metabolic abnormalities and their underlying etiology will help to define diet and 

physical activity guidelines for preventing and treating MetS — an important 

aspect of CVD prevention. 

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15 

CONTENTS 

Dietary Fatty Acids and 



Introduction: Dietary Fat Intake, Inflammation, and Metabolic 

Syndrome —Double Hit Hypothesis ......................................................243 

Dietary Fatty Acids, Inflammation, and Insulin Signalling: 

Cellular Perspective.................................................................................244 

Whole Body Metabolic Perspective: Evidence from Human 

Dietary Fatty Acid Intervention Studies .................................................246 

Future Perspectives ...........................................................................................248 

References .........................................................................................................248 

INTRODUCTION: DIETARY FAT INTAKE, INFLAMMATION, AND 

METABOLIC SYNDROME —DOUBLE HIT HYPOTHESIS 

Nutrition is a key environmental factor that is particularly involved in the pathogenesis 

and progression of several polygenic, diet-related diseases. Metabolic 

syndrome is a very common condition, characterized by insulin resistance, 

abdominal obesity, dyslipidemia [increased triacylglycerol (TAG) or reduced high 

density lipoprotein (HDL) cholesterol concentrations], hypertension, and urinary 

microalbuminuria. 1–3 It often precedes type 2 diabetes mellitus (T2DM) and is 

associated with a greater risk of cardiovascular disease (CVD). 4,5 

The development of metabolic syndrome can be attributed to both genetic 

and environmental factors, as reviewed elsewhere. 6 Figure 15.1 illustrates that 

insulin resistance is the most important metabolic defect that leads to the development 

of the metabolic syndrome — a state that may be triggered by excessive 

metabolic stressors [high fat diet, obesity, elevated plasma non-esterified fatty 

acid (NEFA) levels, etc.]. 7,8 

The link between dietary fat, obesity, and T2DM is partly due to elevated 

fatty acid concentrations on systemic responsiveness to insulin, resulting in 

impaired insulin action in several peripheral tissues including the liver, skeletal 

muscles, and adipose tissues and dysregulated carbohydrate and lipid metabolism, 

which leads to a compensatory hyperinsulinemia. 9 Interestingly, insulin resistance 

243


Progressive phenotype 

Metabolic 

stress 

Sensitive Genotype 

Obesity 

Insulin resistance 

Glucose intolerance 

Metabolic syndrome 

T 2 DM 

Inflammation 

FIGURE 15.1 Development and progression of metabolic syndrome. (From Roche, H.M., 

Proc Nutr Soc 64, 23, 2005. With permission.) 

often co-exists with a subacute chronic pro-inflammatory state. 10–12 Indeed, recent 

research suggests that pro-inflammatory cytokines derived from adipose tissue 

play a key role in the development of insulin resistance. 13,14 The model in Figure 

15.1 suggests that an individual with a genetic predisposition to metabolic syndrome 

cannot sustain a “double hit” from metabolic and inflammatory stressors. 

Also, the pathway between obesity and insulin resistance toward the metabolic 

syndrome and T2DM represents a progressive phenotype. Therefore, attenuating 

the impacts of metabolic stressors through dietary fat modification to improve 

insulin sensitivity would be advantageous. 

DIETARY FATTY ACIDS, INFLAMMATION, AND INSULIN 

SIGNALLING: CELLULAR PERSPECTIVE 

Several lines of evidence suggest that the combination of excessive nutrientderived 

metabolic stressors and pro-inflammatory stressors plays an important 

role in the development of insulin resistance and metabolic syndrome. High fat 

diets and excessive adipose tissue TAG storage result in increased circulating 

plasma NEFA flux to peripheral tissues that promotes insulin resistance. Fatty 

acids induce insulin resistance through inhibition of insulin signaling. 15–17 

Evidence also indicates that saturated fatty acids (SFAs) may play a particular 

role in attenuating peripheral tissue responsiveness to insulin 3 while some polyunsaturated 

fatty acids (PUFAs) counteract this effect. Adipose tissue may be the 

source of insulin de-sensitization of pro-inflammatory molecules collectively 

known as adipocytokines or adipokines, which predispose to insulin resistance. 

13,14 Tumor necrosis factor (TNF)-α, interleukin (IL)-6, resistin, and adiponectin 

(acrp30) have all been shown to influence insulin sensitivity. 18–20 The 

insulin de-sensitizing effects of TNF-α are probably best characterized. TNF-α 

Molecular mechanisms

Dietary Fatty Acids and Metabolic Syndrome 245 

inhibits autophosphorylation of tyrosine residues of the insulin receptor, promotes 

serine phosphorylation of insulin receptor substrate (IRS)-1, and reduces transcription 

of key targets in the insulin signaling cascade, all of which impede 

transduction of the insulin signal. 21 

Knocking out TNF-α or the TNF-α receptor improves insulin resistance in 

animal models of obesity-induced insulin resistance. 22,23 Other groups have proposed 

that other components of the inflammatory response, IκB kinase-β (IKKβ) 

or c-Jun amino-terminal kinases (JNKs), are central to the interplay of dietary 

fatty acids, obesity, and insulin resistance. 24,25 In summary, these studies suggest 

that down-regulation of several components of the inflammatory response affords 

substantial protection from obesity-induced insulin resistance. 

Consistent with our hypothesis that dietary fatty acids can modulate the 

inflammatory profiles of adipocytes, two recent studies determined the effects of 

other fatty acids in vitro. The first showed that the palmitate SFA activated nuclear 

factor-κB (NF-κB) activity and induced TNF-α and IL-6 expression in 3T3-L1 

adipocytes. 26 The second extensive investigation demonstrated that a mixture of 

SFA and PUFA NEFA treatments impaired insulin signalling at multiple sites, 

decreased insulin-stimulated glucose transporter (GLUT)-4 translocation and glucose 

transport, and activated the stress/inflammatory kinase JNK pathway in 3T3- 

L1 adipocytes. 27 Thus it may be possible to attenuate the pro-inflammatory phenotype 

associated with obesity-induced insulin resistance by altering the composition 

of circulating NEFAs through dietary fatty acid modification. 

Therefore, in terms of manipulating dietary factors to attenuate the inflammatory 

response in adipose tissue to improve insulin sensitivity, the most obvious 

treatment is to reduce adipose tissue mass. Nevertheless the prevalence of obesity 

is increasing and due to poor compliance, current therapies are largely ineffective. 

Therefore other strategies to attenuate the impact of insulin resistance in the 

presence of obesity are required. 

Our group demonstrated that a sub-group of fatty acids known as conjugated 

linoleic acids and, in particular, the cis-9, trans-11 CLA isomer (c9, t11-CLA), 

may have the potential to improve lipid metabolism and insulin sensitivity within 

the context of obesity. 28,29 This effect was ascribed to differential sterol regulatory 

element-binding protein (SREBP)-1c gene expression, a key regulatory transcription 

factor involved in lipogenesis and glucose metabolism. Feeding a c9, t11- 

CLA-rich diet produced divergent tissue-specific effects on SREBP-1c expression, 

significantly reducing hepatic SREBP-1c and increasing adipose tissue 

SREBP-1c expression, both of which could contribute to improved lipid and 

glucose metabolism. 28 Interestingly, this study also showed that TNF-α regulated 

SREBP-1c expression in human adipocytes, but not in hepatocytes, thus supporting 

the hypotheses that crosstalk exists between molecular markers of insulin 

sensitivity and adipocytokines which in turn can be modified by fatty acids. 

Further work showed that the insulin sensitizing effect of the c9, t11-CLArich 

diet was associated with a marked reduction in adipose tissue TNF-α expression 

that may be related to lower NF-κB DNA binding, which has been attributed 

to lower nuclear P65 levels and increased cytosolic inhibitor of κBα (IκBα)


expression. 29 This study suggests that the fatty acid composition of the diet can 

be adjusted to attenuate the pro-inflammatory insulin de-sensitizing effect of 

obesity-induced insulin resistance. Indeed there was a significant reduction in the 

adipose tissue macrophage population observed in the c9, t11-CLA fed mice. 

This is an extremely important observation, in that it shows that dietary modification 

can alter the cellular profile of adipose tissue in obesity, which was 

associated with positive metabolic effects. 

WHOLE BODY METABOLIC PERSPECTIVE: EVIDENCE FROM 

HUMAN DIETARY FATTY ACID INTERVENTION STUDIES 

Several studies have shown a consistent relationship between plasma fatty acid 

composition and insulin resistance. A prospective cohort study investigated the 

interaction between serum fatty acid composition and the development of T2DM 

in a cohort of middle-aged normoglycemic men. 30 Baseline serum esterified and 

non-esterified SFA levels were significantly higher and PUFA levels were lower 

in the men who developed T2DM after 4 years. 

Recent evidence from the Nurses’ Health Study showed that higher intake of 

saturated fat and a low dietary P:S ratio were related to increased CVD risk 

among women with T2DM. 31 This study estimated that replacement of 5% of 

energy from saturated fat with equivalent energy from carbohydrates or MUFAs 

was associated with 22 and 37% lower risks of CVD, respectively. This finding 

suggests that dietary fatty acid modification may also determine secondary outcomes 

associated with metabolic syndrome and T2DM. 

Relatively few human dietary intervention studies have determined the relationship 

between dietary fatty acid composition and insulin sensitivity as the 

primary metabolic end point. For the purpose of this review, studies that investigated 

the effect of isocaloric substitution of dietary fatty acids will be reviewed, 

to exclude confounding effects of reduced energy intake on body weight. The 

KANWU study, a controlled multicenter, isoenergetic dietary intervention study 

involving 162 individuals, showed that decreasing dietary SFA and increasing 

MUFA improved insulin sensitivity but had no effect on insulin secretion. 32 

Interestingly the favorable effect of substituting SFA for MUFA was only seen 

when total fat intake was below 37% energy. Within each dietary group a second 

assignment of n-3 PUFA supplementation or placebo was completed, but the n- 

3 PUFA intervention had no effect on insulin sensitivity despite reduced TAG 

concentrations. 

A smaller randomized crossover dietary intervention study in 59 young 

healthy subjects randomly assigned to isoenergetic carbohydrate- and MUFArich 

diets for 28 days significantly improved insulin sensitivity compared to a 

high-SFA diet. 33 Also ex vivo analysis showed that both the carbohydrate- and 

MUFA-rich diets significantly increased basal and insulin-stimulated glucose 

uptake in monocytes. In contrast, another randomized, double-blind, crossover 

study comparing the effect of MUFA, SFA, and trans-fatty acid diets failed to


show any significant effects on insulin sensitivity or secretion. 34 When the group 

members were subdivided according to body mass index (BMI), insulin sensitivity 

was 24% lower in overweight individuals (BMI >25 kg/m 2 ) after the SFA diet 

compared to the MUFA diet. It is interesting to note that these diets were very 

low in fat (28% energy), Therefore the effects of dietary fat composition may be 

more obvious without the background low fat diet. 

Overall, human dietary intervention studies suggest that the removal of dietary 

saturated fat, as verified by alterations in plasma fatty acid composition, can have 

a direct effect on insulin sensitivity. This effect has also been confirmed in a 

metabolic study that showed that altering the compositions of infused free fatty 

acids affected insulin sensitivity. A SFA-rich lipid infusion significantly reduced 

insulin sensitivity indices (40 to 50%) to a much greater extent than a PUFA-rich 

lipid infusion (20 to 27%) in healthy subjects. 35 

Recent studies have attempted to determine whether alterations in dietary fat 

intake or endogenous fatty acid synthesis accounted for altered fatty acid composition 

associated with metabolic syndrome. Cross-sectional data suggest that 

insulin-resistant states are associated with high levels of activity of stearoyl-CoA 

desaturates (SCD-1) and Δ6-desaturase (D6D) and low Δ5-desaturase (D5D) 

activity. 36 

In a prospective study, Warensjo et al. 37 evaluated serum cholesteryl ester 

fatty acid composition and estimated SCD-1, D6D, and D5D activities as precursors 

to fatty acid ratios. The study showed that baseline fatty acid profiles 

predicted the development of metabolic syndrome 20 years later, SFA levels were 

significantly higher and linoleic acid levels were lower in subjects who subsequently 

developed metabolic syndrome by age 70. In addition SCD-1 and D6D 

activities were significantly higher and D5D activity was lower in those who 

developed metabolic syndrome during follow-up. The clinical relevance of altered 

desaturase activity in the development of the metabolic syndrome requires further 

study. 

Long chain n-3 PUFAs have a number of positive health benefits relevant to 

metabolic syndrome, particularly with respect to TAG metabolism. 36 However, 

we have relatively little evidence that n-3 PUFA supplementation improves insulin 

sensitivity in humans despite several studies showing that feeding n-3 PUFA had 

positive effects on glucose and insulin metabolism in different animal models of 

T2DM and metabolic syndrome. 39,40 

Also human epidemiological studies suggest that habitual dietary fish intake 

is inversely associated with the incidence of impaired glucose tolerance and 

T2DM. 41,42 Some studies have reported positive effects of n-3 PUFA supplementation 

on insulin sensitivity in individuals with impaired glucose tolerance and 

diabetes. 43,44 Other studies have not shown positive effects 32,45 even though n-3 

PUFA supplementation improved TAG metabolism. Clearly the putative effects 

of n-3 PUFA on human insulin resistance and impaired glucose tolerance require 

further clarification.


FUTURE PERSPECTIVES 

The increasing prevalence of obesity requires us to reduce the impact of the 

adverse health effects, particularly T2DM and CVD. By 2010, some 31 million 

people in Europe and an estimated 239 million worldwide will require treatment 

for T2DM and its related complications. 46,47 The incidence of the metabolic 

syndrome is increasing exponentially as a consequence of the sharp rise in obesity 

— the key etiological factor in the development and severity of metabolic syndrome. 

To date, public health strategies have been largely unsuccessful at reducing 

the prevalence of obesity. Therefore dietary interventions that attenuate the severity 

of metabolic syndrome within the context of obesity are required and attenuating 

the impact of environmental causes through dietary fatty acid modification 

to improve insulin sensitivity would be advantageous. 

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16 

CONTENTS 

Lipid-Induced Death of 

Macrophages: 

Implication for 

Destabilization of 

Atherosclerotic Plaques 

Oren Tirosh and Anna Aronis 

Abstract .............................................................................................................251 

Introduction .......................................................................................................252 

Macrophage Cell Death ....................................................................................253 

Programmed Cell Death..........................................................................253 

Cell Death and Atherosclerosis...............................................................254 

Effects of TGs on Macrophage Cell Deaths...........................................255 

Potential Therapies............................................................................................258 

References .........................................................................................................258 

ABSTRACT 

In recent decades, the incidence of atherosclerosis in Western society has been 

on the rise. Lipid particles, of which low density lipoprotein (LDL) is the most 

studied, accumulate in the intima of blood vessels and lead to inflammatory 

processes. Leukocytes, mostly macrophages, are recruited from circulating blood 

to the vessel walls. After they are overexposed to lipids and transform to foam 

cells, cell death processes take place. In macrophages, the exposure to lipids is 

known to trigger cell deaths of both apoptotic and necrotic types. External, 

receptor-dependent, and internal mitochondria-affecting apoptotic death pathways 

are involved in these processes. Although oxidized LDL is known as a 

major factor for plaque formation, triacylglycerols (TGs) are increasingly 

believed to be independently associated with coronary heart disease. Accumulation 

of intracellular TGs causes elevation of reactive oxygen species (ROS), 

251


probably of mitochondrial sources, and cell death. In this chapter, the role of 

macrophage cell death in atherogenic plaque instability and the specific effects 

of TG in macrophage lipotoxicity are discussed. 

INTRODUCTION 

Coronary heart disease (CHD) is the largest cause of morbidity and mortality in 

the Western world. 1 Three cellular components of the circulation, monocytes, 

platelets, and T lymphocytes, together with two cell types of the artery wall cells, 

endothelial and smooth muscle cells (SMC), interact in multiple ways in concert 

with lipoprotein particles in generating atherosclerotic lesions. Accumulation and 

oxidation of LDL in vessel walls promotes up-regulation of adhesion molecules 

in endothelial cells and leads to recruitment of blood monocytes and lymphocytes 

to the intima. 2 

As shown by epidemiological and clinical studies, the Western way of life 

and consumption of an unbalanced diet lead to a high incidence of atherosclerosis 

and conditions significantly increasing the risk of CHD such as obesity, metabolic 

syndrome, and diabetes. 3 An atherogenic shift in blood lipid spectrum profile that 

may manifest by increases of serum levels of cholesterol, LDLs, TGs, and very 

low density lipoproteins (VLDLs), and decreases of serum levels of anti-atherogenic 

HDLs has become a frequent problem in developed countries. 

Recruitment of immune cells to the intima of blood vessels indicates an 

inflammation reaction caused by lipid particles. Indeed, obese persons have 

increased serum or plasma concentrations of acute-phase proteins or pro-inflammatory 

cytokines such as C-reactive protein (CRP), interleukin (IL)-6, IL-8, and 

tumor necrosis factor (TNF). Circulating levels of these immune mediators can 

be lowered by weight loss 4,5 or other plasma lipid-lowering interventions, for 

example, short-term diet and exercise. 6,7 CRP, which has been reported to be 

expressed by the liver, macrophages and SMC-like cells, correlates with macrophage 

accumulation in coronary arteries. 8 Therefore, a reduction of CRP by lipidlowering 

measures can contribute to prevention of macrophage accumulation in 

atherosclerotic plaques. 

LDL cholesterol is currently defined as a major risk factor of CHD. However, 

the role of TG, which is increasingly believed to be independently associated 

with CHD, is not yet well studied. A high blood level of TG often occurs together 

with low HDL. Such an effect on the blood lipid profile often occurs with normal 

levels of LDL-C. These abnormalities of the TG-HDL axis are characteristic of 

patients with metabolic syndrome. 9 Recent clinical studies have also revealed that 

increased serum triglyceride (TG) levels are closely related to atherosclerosis 

independently of serum levels of HDL and LDL. 

Among TG-rich lipoproteins (TRLs), remnant lipoproteins (RLPs) are considered 

atherogenic and independent coronary risk factors. It was previously 

reported 10 that monocytes cultured in the presence of RLPs increased their adhesion 

capability to vascular endothelial cells. In the Third Report of the Adult 

Treatment Panel (ATP III) establishing the National Cholesterol Education

Lipid-Induced Death of Macrophages 253 

Program (NCEP) in the United States, the attitude toward elevated plasma triglyceride 

levels has changed, especially as related to moderately elevated and 

borderline high levels. TGs were cited as independent risk factors for CHD. 

PROGRAMMED CELL DEATH 

MACROPHAGE CELL DEATH 

Apoptosis is often associated with morphological and biochemical changes. 11,12 

During apoptosis, the nucleus and cytoplasm are condensed and the dying cells 

disintegrate into membrane-bound apoptotic bodies. Nucleases are activated and 

cause the degradation of chromosomal DNA, at first into large chromosomal 

DNA (50 to 300 kb) and ultimately into very small oligonucleosomal fragments. 

11,12 The signaling events leading to apoptosis can be divided into two 

distinct pathways involving either mitochondria or death receptors. 13,14 

In the mitochondrial pathway, death signals led to changes in mitochondrial 

membrane permeability and the subsequent release of pro-apoptotic factors 

involved in various aspects of apoptosis. 13 The released factors included cytochrome 

c (cyt c), 15 apoptosis inducing factor (AIF), 16 second mitochondriaderived 

activator of caspase (Smac/DIABLO), 17,18 and endonuclease G. 19 Cytosolic 

cyt c forms an essential part of the apoptosome apoptosis complex composed 

of cyt c, Apaf-1, and procaspase-9. Formation of the apoptosome leads to the 

activation of caspase-9, which then processes and activates other caspases to 

orchestrate the biochemical executions of cells. Smac/DIABLO is also released 

from the mitochondria along with cyt c during apoptosis, and it functions to 

promote activation of caspases by inhibiting IAP (inhibitor of apoptosis) family 

proteins. 17,18 

In the death receptor pathway, the apoptotic events are initiated by engaging 

the tumor necrosis factor (TNF) family receptors including eight different death 

domain (DD)-containing receptors (TNFR1 also called DR1; Fas, also called 

DR2; DR3, DR4, DR5, DR6, NGFR, and EDAR). 20,21 Upon ligand binding or 

when overexpressed in cells, DD receptors aggregate, resulting in the recruitment 

of various adapter proteins that mediate both cell death and proliferation 20,21 and 

can activate the caspase system. The human genome encodes 12 to 13 distinct 

caspases that function in cytokine processing and inflammation, and at least seven 

(caspases 2, 3, 6, 7, 8, 9, and 10) contribute to cell death. 22 To date more than 

280 caspase targets have been identified. 23 Some of these proteins may be cleaved 

very late and less completely during apoptosis or may not be cleaved in all cell 

types. The functional consequences of the cleavage of many of the identified 

substrates are unknown. 23 Most of the protein substrates can be categorized into 

a few functional groups: apoptosis regulator, cell adhesion, cytoskeletal and 

structural, etc. Rucci et al. noted that proteins of the electron transfer chains of 

mitochondria are targets for caspase-dependent degradation during apoptosis. 24 

Active caspase-9 and caspase-3 have been observed in the mitochondria, but 

their origins are unclear. Theoretically, procaspase-9 may be activated in the


mitochondria in a cytochrome c/Apaf-1-dependent manner, or activated caspase- 

9 and -3 may translocate to the mitochondria as suggested by Chandra and Tang. 25 

Using a system of positive staining of the mitochondria with calcein and cobalt 

as a fluorescent quencher, Poncet et al. showed that during induction of apoptosis, 

the inner membrane of the mitochondria losses its barrier function and becomes 

permeable. 26 These data suggest that caspase activity can translocate to the mitochondrial 

matrix. Indeed strong support for this idea is based on detection of 

mitochondrial caspase activity in real time, in situ, in live cells. Using a mitochondrially 

targeted CFP-caspase 3 substrate-YFP construct (mC3Y), a caspase- 

3-like activity in the mitochondrial matrices of some cells during apoptosis was 

demonstrated. 27 

CELL DEATH AND ATHEROSCLEROSIS 

One of the earliest events in atherosclerosis is the entry of monocytes into focal 

areas of the arterial subendothelium. Once inside the arterial intima, monocytes 

differentiate into macrophages. 28 Macrophages are constantly exposed to excess 

lipids following overnutrition and they are also involved in the development of 

atherosclerosis — an inflammatory disease process. 29–31 Macrophages are considered 

to constitute one of the most important factors in its initiation and progression. 

Macrophages accumulate large amounts of intracellular cholesterol via 

accumulation of lipoproteins. The presence of cholesterol-loaded macrophages 

in atherosclerotic lesions is a prominent feature throughout the lives of the lesions 

and these cells exert major impacts on lesion progression. 

Dead macrophages are frequently observed in human atherosclerotic lesions, 

and are considered to be involved in atherosclerotic plaque instability. 32 Unstable 

plaques demonstrate a greater portion of apoptotic cells than stable ones. 33,34 Immunohistochemical 

staining of ruptured plaques has shown that apoptotic nuclei in 

plaque rupture sites are essentially those of macrophages and much less frequently 

of smooth muscle and T lymphocytes. 33 Fibrous caps of ruptured plaques have more 

macrophages and also contain fewer SMCs than those of unruptured ones. The 

death of macrophages and SMCs in blood vessels is a complicated process owing 

to their mutual interactions and the presence of monocytes and macrophages in 

plaques increases also the rate of SMC apoptosis. 35 

Accumulated evidence suggests that plaque macrophage deaths are due to 

three possible pathways: (1) Fas-mediated death and promotion of inflammation, 

(2) oxidized LDL which at least in vitro was proven to be an effective cell death 

inducer following cellular uptake, and (3) excess accumulation of free cholesterol 

that may lead to caspase-dependent or independent cell death. The level of 

apoptotic cell death is strongly related to the stage of development of the atherosclerotic 

plaque. 36 It has been suggested that in the early stages of lesions, the 

induction of macrophages apoptosis actually prevents the growth and development 

of the lesion. This effect is probably attributed to controlling the number 

of macrophages in the lesion. The experimental evidence of this effect of apoptosis 

includes a number of genetic alterations in mouse models of atherosclerosis


that resulted in early increases or decreases of macrophage apoptosis. An inverse 

relationship was found between early lesional macrophage apoptosis and lesion 

area. 

When bone marrow taken from P53–/– (a pro-apoptotic protein) mice was 

transplanted to APOE*3-Leiden mice, a significant 2.3-fold increase was 

observed in early lesion size. When bone marrow of Bax–/– mice was transplanted 

to LDLR–/– mice, the same effect was observed, indicating a negative 

effect of apoptosis on plaque growth. On the other hand, in an advanced atherosclerotic 

plaque region, the apoptotic processes may lead to massive macrophage 

cell deaths. Impaired capacities of other macrophages to clean and clear 

the plaque regions from cell debris may lead to the development of necrotic 

cores and accelerated inflammatory processes in the intima. Macrophages 

express multiple metalloproteinases (e.g., stromelysin) and serine proteases 

(e.g., urokinase) that degrade the extracellular matrix, weakening the plaque and 

making it rupture-prone. 

Most studies report apoptotic macrophage deaths in ruptured plaques on the 

basis of immunohistochemical staining methods. However, standard TUNEL 

DNA staining lacks specificity, especially in late phases of cell death when most 

DNA passes degradation. 36 Moreover, because of variable uptake of lipids and 

differences in cell size, apoptosis-characterizing cell shrinkage is not always 

apparent. 33 This is why an indication of the active form of “committing-to-die” 

caspase-3 is necessary for final determination of apoptotic cell death. Some 

researchers indicate caspase-3 independent cell deaths in LDL-exposed macrophage 

cell cultures or histological cuts of plaques, 33,37 supposing an alternative 

of oncotic (necrotic) cell death. 

EFFECTS OF TGS ON MACROPHAGE CELL DEATHS 

Previous studies have suggested that TGs are the main signaling components of 

endocytosed VLDL in macrophages. 38 Our study showed death pathways in 

macrophages resulting from exposure to TGs — a mechanism that may be relevant 

to the development of atherosclerosis. Murine J774.2 macrophages in culture 

were exposed to a commercial soybean oil-based TG lipid emulsion (0.1 to 1.5 

mg lipid/ml) known to be taken up by macrophages via a coated pit-dependent 

mechanism mediated by macrophage secretion of apolipoprotein E. 40 The exposure 

of the cells to the lipid emulsion led to intracellular change in fatty acid 

profile and high accumulation of TGs as measured with gas chromatography and 

Nile red staining, respectively (Figure 16.1). The TG effect culminated in cell 

death, with no caspase-3 activation. Dual staining with propidium iodide and 

annexin V followed by flow cytometric analysis showed that TG facilitated cell 

deaths with clear necrotic characteristics. 

Reactive oxygen species (ROS) are involved in macrophage activation and 

death. While ROS may induce macrophage activation, macrophage foam cells 

contain potent oxidant-generating lipid-targeting systems such as inducible NO


Events 

128 

C 

12h 

0 

10 

Nile Red fluorescence 

0 101 102 103 104 FIGURE 16.1 Accumulation of intracellular lipids following exposure of J774.2 macrophages 

to lipid treatment. Nile red staining of the cells was carried out after treatment 

with soybean oil-based lipid emulsion, 1 mg/ml. 

synthase and 15-lipoxygenase, allowing increased recognition and uptake by 

macrophages and creating a positive feedback loop. 41 

Following 24 hr of exposure of macrophages to 1 mg/ml TGs, cellular ROS 

levels were strongly elevated. In contrast, after 48 hr, when 50% of the macrophages 

underwent cell death, ROS production was arrested. Most of the TGmediated 

ROS production was demonstrated to be via mitochondrial complex 1 

of the electron transfer chain, as demonstrated by the use of rotenone, a mitochondrial 

complex 1 inhibitor that significantly attenuated cellular ROS levels in 

TG-treated cells (Scheme 16.1). 

To elucidate whether the cell death process was indeed oxidant dependent, 

antioxidant protection was studied. Treatment with 0.5 mM N-acetyl-cysteine 

(NAC), 0.05 mM ascorbic acid, and 0.2 mM resveratrol protected against the TG 

lipotoxic effect, while lipophilic antioxidants surprisingly did not. For further 

study, the combination of NAC, ascorbic acid, and resveratrol was used at much 

lower concentrations (one-tenth of original concentrations), which led to the 

appearance of a synergistic protective effect. 

Exposure of J774.2 macrophages to increased levels of TG leads to the 

induction of oxidative stress-mediated lipotoxicity. Decreased GSH levels and 

the protective role of NAC are strong indications of the pivotal role of ROS in 

TG-induced lipotoxicity. In our model, the NAC, ascorbic acid, and resveratrol 

antioxidants played a protective role in TG-induced lipotoxicity. The protective 

effect of NAC, which is a precursor of glutathione, suggests an interaction in the 

level of water-soluble antioxidants. Moreover, TG caused ROS-dependent G1 

arrest in the macrophage cell cycle. The exposure of untreated cells to an inhibitor 

of glutathione synthesis BSO mimicked TG-induced G1 arrest, reinforcing an 

important role of GSH in prevention of TG-induced mechanisms of lipotoxicity. 

24h 

48h


Extra cellular 

Intra cellular 

TG particle 

ROS 

Caspase-3 

GSH 

Cell death Necrosis 

SCHEME 16.1 Mechanism for TG-induced cell death in macrophages. Lipid droplets 

accumulate inside the macrophages, in time generating foam cells. Oxidative stress develops 

as a result of the lipid stress and leads to a necrotic type of cell death. Overall, the 

effect of macrophage disintegration in atherogenic plaques will result in inflammation and 

plaque rupture due to destabilization. 

It is possible that prolonging ROS production from endogenous cellular 

sources such as mitochondria may attenuate caspase activity and shift apoptosis 

to necrosis. We therefore evaluated the effect of TG on apoptotic cells showing 

high caspase activity. Indeed, TG induced elevated ROS levels and suppressed 

caspase-3 in apoptotic cells pretreated for 24 hr with cycloheximide. These results 

indicate that exposure to TG can directly regulate lipotoxicity in macrophages 

by inducing mitochondria-mediated prolonged oxidative stress; this, in turn, can 

inactivate the apoptotic caspase system, resulting in necrotic cell death which can 

be prevented by specific antioxidants. 

Accumulation of TG in macrophages is a unique phenomenon of these cells 

since they are capable of taking up large amounts of this type of lipid. We 

suggest that rapid accumulation of fat may result in oxidative stress-dependent 

suppression of the caspase system which may suppress activation of type 1 

programmed cell death and affect other mechanisms related to cellular signal 

transduction and cell cycle arrest. The TG treatment may promote characteristics 

of necrotic cell death or caspase-independent types of programmed cell 

death. This, in fact, may result in disintegration of lipid-loaded macrophages 

(foam cells) and release of their enzymatic cytosolic and lysosomal contents 

to their surroundings, leading to lesion erosion and rupture of the plaques with 

the formation of a thrombus. 

? 

Mitochondria 

Inflammation


POTENTIAL THERAPIES 

One problem that must be encountered when considering novel therapies in 

atherosclerosis is the heterogeneity of plaque status. In human patients, it is most 

probable that advanced plaques and early plaques are relatively abundant. Therefore, 

a careful approach should be considered. It is recommended that apoptosis 

will be promoted in the early plaques while in advanced plaques where inflammation 

is already a considerable factor, apoptosis should be inhibited. 42 

One approach for blocking macrophage cell death is to use specific antioxidants. 

We found that water-soluble antioxidants are more efficient than lipidsoluble 

antioxidants in preventing TG-induced lipotoxicity in macrophages. 

Therefore the use of antioxidants should be considered according to their relative 

death-preventing effects. Only compounds that may affect lipotoxicity should be 

used. Another approach is to enhance the phagocytic activities of macrophages 

in atherosclerotic plaques. This approach will help to maintain phagocytic activities 

in advance plaques and will prevent the formation of necrotic cores and the 

inflammatory responses. Use of eicosanoids including LXA4, LXB4, and aspirintriggered 

15-epi-LXB4, and their stable analogues stimulated macrophages to 

phagocytose apoptotic neutrophils. Apoplipoprotein E3 can correct defects in 

macrophage phagocytic effects and finally the use of glucocorticoids may boost 

phagocytic activity. 42 

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17 

CONTENTS 

α-Lipoic Acid Prevents 

Diabetes Mellitus and 

Endothelial Dysfunction 

in Diabetes-Prone 

Obese Rats 

Woo Je Lee, Ki-Up Lee, and Joong-Yeol Park 

Introduction .......................................................................................................261 

α-Lipoic Acid....................................................................................................262 

α-Lipoic Acid Prevents Diabetes Mellitus in Diabetes-Prone 

Obese Rats...................................................................................263 

α-Lipoic Acid Prevents Endothelial Dysfunction in Diabetes-Prone 

Obese Rats...................................................................................265 

Summary ...........................................................................................................267 

Acknowledgment...............................................................................................267 

References .........................................................................................................267 

INTRODUCTION 

Recent evidence suggests that oxidative stress plays a causative role in many 

chronic diseases such as diabetes and cardiovascular disease. Many efforts have 

been made to prevent the development and progression of diabetes and cardiovascular 

disease by using materials with antioxidative properties. Among a variety 

of antioxidants, α-lipoic acid (α-LA) has recently received much research attention. 

This chapter will focus on the roles and mechanisms of α-LA in the 

prevention of diabetes mellitus and endothelial dysfunction in obese animals. 

261


α-LIPOIC ACID 

α-LA is a naturally occurring compound that is widely distributed in plants and 

animals. In humans, α-LA can be supplied by diet or obtained through de novo 

synthesis by the liver and other tissues. Only the R-isomer of α-LA is synthesized 

naturally. Conventional chemical synthesis of α-LA results in a 50/50 (racemic) 

mixture of two optical isomers, R-α-LA and S-α-LA. 

Free α-LA is rapidly taken up by cells and reduced to dihydrolipoic acid 

(DHLA) intracellularly. In most cells containing mitochondria, α-LA is reduced 

by an NADH-dependent reaction with lipoamide dehydrogenase to form DHLA. 

In cells that lack mitochondria, α-LA can be reduced to DHLA via NADPH with 

glutathione and thioredoxin reductase. 1 

α-LA contains two sulfur molecules that can be oxidized or reduced (Figure 

17.1). This feature allows α-LA to function as a cofactor for several important 

enzymes as well as a potent antioxidant. α-LA functions as a cofactor of mitochondrial 

key enzymes such as pyruvate dehydrogenase and α-keto-glutarate 

dehydrogenase and thus has been shown to be required for the oxidative decarboxylation 

of pyruvate to acetyl-CoA, the critical step leading to the production 

of cellular energy (ATP). 2,3 In addition, when large amounts of free α-LA are 

available (e.g., with supplementation), α-LA is also able to function as an antioxidant. 

4 α-LA and its reduced form, DHLA, are powerful antioxidants. 5 DHLA 

can then be transported easily out of the interiors of cells and function effectively 

in the extracellular spaces. Thus, in this form, it is believed to function directly 

as an antioxidant and possess its greatest antioxidant potential. 6 However, because 

DHLA is also rapidly eliminated from cells, the extents to which its antioxidant 

effects can be sustained remain unclear. 

Many reports describe the functions of α-LA 4,5,7–10 and the functions include 

(1) quenching of reactive oxygen species, (2) regeneration of exogenous and 

endogenous antioxidants such as vitamins C and E and glutathione, (3) chelation 

of metal ions, and (4) prevention of membrane lipid peroxidation and reparation 

of oxidized proteins. Because of its antioxidant properties, α-LA is known to be 

effective in both prevention and treatment of oxidative stress in a number of 

Lipoic acid 

Dihydrolipoic acid 

(DHLA) 

S — S 

FIGURE 17.1 Structures of lipoic acid and dihydrolipoic acid. 

∗ 

SH SH 

∗ 

O 

O 

OH 

OH

Diabetes Mellitus and Endothelial Dysfunction 263 

models or clinical conditions including diabetes, 11–14 vascular dysfunction, 15 and 

neurodegenerative diseases. 16 

α-LIPOIC ACID PREVENTS DIABETES MELLITUS IN DIABETES-PRONE 

OBESE RATS 

Increased oxidative stress is a widely accepted participant in the development 

and progression of diabetes and its complications. 17–19 Diabetes is usually accompanied 

by increased production of free radicals 18–21 or impaired antioxidant 

defenses. 22,23 Hence, it is likely that a substance known to reduce oxidative stress 

would reduce the progression of cell damage in diabetes. 

α-LA, an essential cofactor in oxidative metabolism, has been reported to 

exert a number of potentially beneficial effects in oxidative stress-related conditions. 

Experimental and clinical studies have indicated that α-LA treatment 

reduces the development of diabetic complications. 7,24–29 Furthermore, α-LA 

facilitates glucose metabolism and increases glucose uptake leading to improved 

glucose utilization in vitro and in vivo. 30 These features may enable α-LA to be 

used as a potential agent for the prevention and treatment of diabetes mellitus. 

Experimental and clinical studies have indicated that α-LA improved insulin 

sensitivity. α-LA administration improved insulin-stimulated whole body and 

skeletal muscle glucose utilization in insulin-resistant obese rats 30,31 and type 2 

diabetic humans. 32,33 Moreover, several studies using cultured muscle cells, 34 

isolated rat diaphragm, 35 and perfused preparations of normal and diabetic 

rabbits 36 demonstrated that α-LA activates glucose transport and stimulates 

glycolysis. 

Obesity is the most important risk factor for type 2 diabetes mellitus. 37 Many 

possible mechanisms link obesity and the development of diabetes mellitus. One 

mechanism is that oxygen free radicals derived from excessive triglycerides and 

long chain fatty acyl CoA (LCAC) in muscles and pancreatic β cells cause 

functional defects in these tissues, leading to the development of type 2 diabetes. 

38–40 α-LA has been shown to protect against oxidative stress-induced insulin 

resistance in vitro 41,42 and improve insulin sensitivity in human and animal models. 

30,43–47 In addition, it was recently reported that administration of α-LA to 

rodents reduced body weight by suppressing food intake and visceral fat mass. 48 

Thus, it is conceivable that α-LA may have a preventive role in the development 

of diabetes. 

A recent study (14) demonstrated that α-LA could prevent the development 

of diabetes in an obese animal model. The authors used Otsuka Long-Evans 

Tokushima Fatty (OLETF) rats as obese animal models, because these rats are 

obese from a young age and become diabetic after 18 weeks of age. 49 These 

features in OLETF rats resemble those of human type 2 diabetes. To investigate 

the preventive effect of α-LA on the development of diabetes, 9-week old OLETF 

rats were fed standard rat chow with or without racemic α-LA (200 mg/kg of 

body weight/day) for 3 weeks and the development of diabetes was evaluated. 

Approximately 80% of rats fed rat chow without α-LA developed diabetes at 40


weeks of age, but none of the rats fed rat chow with α-LA developed diabetes. 

In addition, administration of α-LA protected pancreatic β cell destruction and 

reduced triglyceride accumulations in skeletal muscles and pancreatic β cells. 

These data indicate that α-LA prevents the development of diabetes in diabetesprone 

obese rats by decreasing lipid accumulation in skeletal muscle and exerting 

beneficial effects on pancreatic β cells. 

This report seems to indicate that the mechanism of preventive effect of α- 

LA on the development of diabetes may be multifarious. First, because oxidative 

stress has been suggested to be associated with insulin resistance 50,51 and α-LA 

has potent antioxidative capacities, its preventive effects may be due to its antioxidant 

action. Indeed, α-LA reduced plasma levels of oxidative stress markers 

such as malondialdehyde and 8-hydroxy-deoxyguanosine. However, this relationship 

between protective effect and antioxidant action of α-LA was not investigated 

directly. 

A second possible mechanism of the preventive role of α-LA is its effects 

on lipids in skeletal muscles and pancreatic β cells. Skeletal muscle is the major 

tissue responsible for 70 to 80% of whole body glucose uptake. Indeed, insulin 

resistance in skeletal muscle is a common characteristic of type 2 diabetes, and 

many previous studies in animal models and human subjects have shown that 

lipid accumulation in skeletal muscle is associated with insulin resistance in 

obesity. In addition, lipid accumulation in pancreatic β cells also affects β cell 

damage. LCAC accumulation in pancreatic β cells may induce apoptosis of the 

cells. Increased plasma free fatty acid by lipid–heparin infusion induced β cell 

hypertrophy. Since α-LA reduces visceral fat mass and decreases plasma free 

fatty acid and triglyceride concentrations and tissue triglyceride contents in 

OLETF rats, insulin resistance in skeletal muscle and β cell apoptosis can be 

prevented. 

While the possible mechanisms of the effects of α-LA on the development 

of diabetes in obese rats could be inferred from this report, we cannot exclude 

the possibility that α-LA prevented the development of diabetes due to its weight 

reducing effects. In addition, the exact molecular mechanism by which α-LA 

reduces lipid contents in skeletal muscle was not investigated in this report. Thus, 

other studies 15,52 were performed to minimize the effect of α-LA on body weight 

change and investigate the mechanism of α-LA in reducing lipid content in 

skeletal muscle. The authors fed rats for a short period (200 mg/kg body weight) 

via intravenous infusion for 2 hr or 0.5% (wt/wt) racemic α-LA (mixed in food 

for 3 days) to minimize the effect of α-LA on body weight change. In addition, 

since AMPK was known to be responsible for the effect of α-LA in the hypothalamus 

and because it increases both glucose uptake and fatty acid oxidation, 

the authors focused on AMPK as a molecular target of α-LA. AMPK is known 

as a cellular “energy sensor” because its activity is sensitively changed by its 

cellular energy state. AMPK controls a number of metabolic processes to 

help restore energy depletion in peripheral tissues. 53,54 For example, AMPK activation 

in skeletal muscle enhanced glucose uptake and mitochondrial fatty acid


oxidation. 55 AMPK is also expressed in vascular endothelium, 56 and AMPK 

dysregulation has been suggested to contribute to endothelial dysfunction. 57 

Administration of α-LA to OLETF rats improved insulin-stimulated whole 

body glucose uptake and whole body glycogen synthesis. Insulin-stimulated 

glucose uptake and glycogen synthesis in skeletal muscle were also improved in 

OLETF rats treated with α-LA. In skeletal muscles of OLETF rats, AMPK (α2- 

AMPK, not α1-AMPK) activity was decreased compared to control rats. The 

reason for the decreased AMPK activity is not yet known. However, because 

AMPK is enzyme activated when the cellular energy state is low, abundant fuels 

such as elevated plasma free fatty acid and/or glucose in OLETF rats may reduce 

AMPK activity. 

Administration of α-LA increased α2-AMPK and fatty acid oxidation and 

decreased triglyceride contents in skeletal muscles of OLETF rats. Overexpression 

of the dominant negative α2-AMPK gene in skeletal muscles of OLETF rats 

reversed the effects of α-LA on triglyceride contents, fatty acid oxidation, and 

insulin-stimulated glucose uptake, suggesting that α-LA exerted its effects via 

AMPK activation. Because AMPK is known to increase fatty acid oxidation and 

because lipid accumulation in muscle is associated with insulin resistance, we 

could deduce from this report that α-LA improved insulin sensitivity by activating 

AMPK and increasing fatty acid oxidation, and subsequently by reducing lipid 

accumulation in skeletal muscle. However, further study is necessary to explain 

the precise molecular mechanism by which α-LA prevents the development of 

diabetes and improves insulin sensitivity. 

α-LIPOIC ACID PREVENTS ENDOTHELIAL DYSFUNCTION IN DIABETES- 

PRONE OBESE RATS 

Oxidative stress and impaired bioactivity of vascular nitric oxide (NO) play 

important roles in the pathogenesis of microvascular and macrovascular complications 

in diabetes mellitus. Antioxidative properties and the capacity of increasing 

NO synthesis and activity may contribute to the protective role of α-LA in 

the development of vascular complications in diabetes. It was reported that O 2 – 

production was increased in aortic tissues of insulin-resistant rats, 43,58 and α-LA 

supplementation in chronically glucose-fed rats prevents the increase in aortic 

basal O 2 – production. In addition, α-LA treatment prevented increases of thiobarbituric 

acid-reactive substances (indirect evidence of intensified free radical production 

and uniformly increased substances in streptozotocin (STZ)-induced diabetic 

rats). 27,28 

α-LA improved NO-mediated vasodilation in diabetic patients but not in 

healthy control subjects. This effect seems to be associated with antioxidative 

properties of α-LA because the effects of α-LA were positively related to plasma 

levels of malondialdehyde. 59 In an in vitro study, however, α-LA increased NO 

synthesis and bioactivity in human aortic endothelial cells by mechanisms that 

appear to be independent of antioxidative properties, because cellular GSH levels 

and GSH:GSSG ratios were not significantly changed by α-LA treatment. 60


α-LA improved impaired endothelium-dependent vasorelaxation in diabetic 

rats. 61 In STZ-induced diabetic rats, hyperglycemia induces decreased eNOS 

expression and increased iNOS expression in tissues such as heart, aorta, sciatic 

nerve, and kidney. α-LA treatment increased eNOS expression and decreased 

iNOS expression. 62 In addition, the decline in eNOS phosphorylation — a possible 

mechanism involved in vascular dysfunction in aging — can be partially restored 

by treating old rats with α-LA. 63 

Moreover, α-LA treatment inhibited adhesion molecule expression in HAEC 

and TNF-α-induced monocyte adhesion. 64 α-LA also inhibited NF-κB-mediated 

transcription and expression of endothelial genes such as tissue factor and endothelin-1 

65 and reduced advanced glycation end product (AGE)-induced endothelial 

expression of VCAM-1 and monocyte binding to the endothelium. 66 

In addition to these mechanisms, α-LA may exhibit diverse protective effects 

in vascular disease. It may lower blood pressure in rats. α-LA supplementation 

decreased systolic blood pressure in spontaneously hypertensive rats, 67 in high 

salt-treated rats, 68 and in fructose-induced hypertensive WKY rats. 69 Similarly, 

hypertension induced by the addition of a 10% D-glucose drink to their diet was 

attenuated by α-LA in Sprague-Dawley rats. 70 

α-LA has exhibited lipid-lowering capacity. In studies of rabbits 71,72 and 

Japanese quail, 73 α-LA decreased levels of cholesterol and lipoprotein in serum. 

It also reduced serum triglyceride levels in STZ-induced diabetic rats. 24 

α-LA has been shown to decrease LDL oxidation. It was reported that DHLA 

inhibited Cu 2+ -dependent LDL peroxidation by chelating copper in in vitro experiments. 

74 In addition, in human studies, α-LA (600 mg/day) significantly 

increased the lag time of LDL lipid peroxide formation for both copper-catalyzed 

and 2,2′-azobis (2-amidinopropane) hydrochloride (AAPH)-induced LDL oxidation. 

75 

Central obesity is an important risk factor for cardiovascular disease. Endothelial 

dysfunction, generally considered a prerequisite for atherosclerosis, is a 

frequent finding in obesity. A recent report indicates that administration of α-LA 

to patients with metabolic syndrome improved endothelial function 76 but the 

mechanism is not yet known. It was suggested that lipid accumulation in vascular 

tissue and a consequent increase in oxidative stress lead to endothelial dysfunction 

in obesity. 39 

As described in the previous section, α-LA enhances fatty acid oxidation and 

reduces lipid accumulation by activating AMPK in skeletal muscles of obese rats. 

Because α-LA produces these effects and because AMPK is expressed in vascular 

endothelial cells, 77 it is conceivable that α-LA may improve endothelial function 

in obesity by activating AMPK. One recent study 15 demonstrated that endothelium-dependent 

vasorelaxation was impaired and AMPK activity in endothelium 

of obese animals was decreased compared to control (lean) rats. In obese rats, 

α-LA administration improved the impaired endothelium-dependent vasorelaxation 

and increased AMPK activity and phosphorylation independent of the effect 

of α-LA on body weight. In addition, in an in vitro study using human aortic 

endothelial cells (HAECs) treated with a fatty acid (linoleic acid), α-LA prevented


linoleic acid-induced decreases in AMPK phosphorylation. This effect was associated 

with normalization of endothelial apoptosis and ROS generation in the 

presence of linoleic acid. These results suggest that the mechanism of endothelial 

dysfunction in obesity is reduced AMPK activity in endothelial cells, and that α- 

LA may improve endothelial dysfunction in obese rats by activating AMPK in 

endothelial cells. 

SUMMARY 

A growing body of evidence suggests that α-LA exerts beneficial effects on both 

prevention and treatment of oxidative stress in a number of models and clinical 

conditions including diabetes and vascular dysfunction. However, before α-LA 

can be used in clinical practice to prevent diabetes or vascular dysfunction, more 

experimental research to elicit the mechanism and clinical trials to determine the 

proper dose, formula, and route of administration are needed. 


This work was supported by National Research Laboratory grant 

M1040000000804J000000810 from the Ministry of Science and Technology of 

the Republic of Korea. 

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18 

CONTENTS 

Lipoic Acid Blocks 

Obesity through 

Reduced Food Intake, 

Enhanced Energy 

Expenditure, and 

Inhibited Adipocyte 

Differentiation 

Jong-Min Park and An-Sik Chung 

Introduction .......................................................................................................274 

Obesity Related to Fatty Cell Biology, Energy Expenditure, and 

Feeding Regulation..................................................................................274 

Antiobesity Function through Suppression of Hypothalamic AMPK .............277 

Regulation of Energy Expenditure and Food Intake ..............................277 

Role of Hypothalamic AMPK.................................................................278 

Reduction of Food Intake and Increase of Energy Expenditure via 

Suppression of Hypothalamic AMPK.........................................278 

Regulation of Adipocyte Differentiation ..........................................................280 

Inhibition of Adipocyte Differentiation via Mitogen-Activated Protein 

Kinase Pathways......................................................................................281 

MAPK Signaling Pathways Mediate Actions of LA 

on Adipogenesis ..........................................................................281 

Modulation of Auxiliary Transcription Factors in Adipogenesis...........281 

MAPK Signaling Pathways Mediate LA Actions on Cell 

Cycle and Clonal Expansion.......................................................282 

Summary ...........................................................................................................283 


References .........................................................................................................284 

273


INTRODUCTION 

Obesity is rapidly increasing throughout the world, substantially shortening life 

expectancy, and closely correlated with the prevalence of diabetes and cardiovascular 

diseases. Plasma levels of leptin, tumor necrosis factor (TNF)-α and nonesterified 

fatty acids are elevated in obesity and substantially contribute to the 

development of insulin resistance. 1 

Although obesity research including studies of leptin 2 and the leptin receptor 

gene 3 has been extensively pursued for more than two decades, the molecular 

mechanisms of obesity are not yet completely understood. Finding target molecules 

of weight regulatory mechanisms will contribute to the development of safe 

and effective pharmaceuticals for blocking obesity and preventing diabetes and 

cardiovascular diseases. α-lipoic acid (LA) and its reduced dihydrolipoic acid 

(DHLA) are considered antioxidants (Figure 18.1). LA has components of αketo 

dehydrogenases including pyruvate dehydrogenases that catalyze various 

redox-based reactions. 4 Recent studies demonstrated that LA facilitates glucose 

transport and utilization in fully differentiated adipocytes as well as animal models 

of diabetes. 5–7 LA also dramatically reduced rodent weights by suppressing food 

intake and increasing energy expenditures. 8 

Obesity is caused by both hypertrophy of adipose tissue and by adipose tissue 

hyperplasia, which triggers the transformation of pre-adipocytes into adipocytes. 9 

LA decreases hypothalamic AMP-activated protein kinase (AMPK) activity and 

induces preformed weight losses in rodents by reducing food intake and enhancing 

energy expenditures. 8 This chapter will focus on antiobesity mechanisms of 

LA: blocking adipocyte differentiation and reducing hypothalamic AMPK. 

OBESITY RELATED TO FATTY CELL BIOLOGY, ENERGY 

EXPENDITURE, AND FEEDING REGULATION 

Adipose tissue is the major storage organ for surplus energy. It is now clear that 

adipose tissue is a complex and highly active metabolic and endocrine organ. 

S 

S 

* 

COOH 

α-Lipoic acid 

(1,2-dithiolane-3-pentanoic acid) 

Reduction 

(2H + +2e - ) 

SH SH 

FIGURE 18.1 Structures of α-lipoic acid and dihyrolipoic acid. 

E 

E: Lipoamide dehydrogenases 

Glutathione reductases 

Thioredoxin reductases 

* 

COOH 

Dihydrolipoic acid (DHLA)

Lipoic Acid Blocks Obesity 275 

Leptin is a representative adipocyte-derived hormone that signals information 

about the body’s energy status from the adipose tissue to the brain. 

Although it is well known that leptin-deficient ob/ob and leptin receptor 

deficient db/db mice develop severe obesity, leptin deficiency is very rare in 

humans. Most obese individuals have increased plasma leptin concentrations, 

suggesting that they are leptin-resistant. In addition to secreting leptin, adipose 

tissue secretes a variety of bioactive peptides, collectively called adipocytokines, 

including TNF-α, adiponectin, plasminogen activator inhibitor (PAI)-1, interleukin 

(IL)-6, and angiotensinogen. The expression profiles of adipocytokines in 

subcutaneous adipose tissues and those in visceral adipose tissues are different. 

Adiponectin, PAI-1, IL-6, and angiotensinogen are mainly shown in the latter. 

The change in these important adipocytokines by visceral obesity is regarded 

as the cause of the detrimental metabolic effects. Recently, the physiologic role 

of adiponectin has received considerable attention. Adiponectin has been shown 

to reduce insulin resistance and atherosclerotic processes and to increase fatty 

acid oxidation rates. 10 The actions of adiponectin are considered to arise from 

the activation of AMPK through the recently cloned adiponectin receptor. 11 Unlike 

other adipocytokines, plasma adiponectin levels are reduced in accordance with 

body (visceral) fat mass. 10 TNF-α, which increases in obesity and inhibits insulin 

signals, is considered a key factor in the regulation of adiponectin production. 

Body weight results from a balance between food intake and energy expenditure. 

The sympathetic nervous system is activated in response to excess energy 

or a cold environment. Mice with deletions of the genes encoding the three 

adrenergic receptor subtypes 12 developed severe obesity as a result of their 

inability to increase energy expenditure in response to a high caloric diet. In 

rodents, uncoupling protein (UCP)-1 in brown adipose tissue (BAT) is the main 

regulator of basal energy expenditure and the expression of this protein is 

increased by adrenergic stimulation. In humans, however, the main regulator of 

energy expenditure is not yet known. The UCP-1 homologues known as UCP-2 

and UCP-3 that are expressed in human tissues were formerly thought to be the 

main regulators of energy expenditure. Indeed, hyperexpression of UCP-2 and 

UCP-3 was shown to increase energy expenditure and reduce body fat. These 

proteins, however, may not be the major regulators of whole body energy expenditure, 

inasmuch as mice deficient in either protein did not develop obesity. 13 

The neural system that regulates body weight and appetite is centered in the 

hypothalamus, which coordinates both afferent sensing and efferent action signals. 

Long-term afferent signals such as leptin and insulin sense the long-term 

status of body energy stores, whereas short-term (meal-related) afferent signals 

derived from the gut are involved in regulating the onset or termination of 

individual meals. Neuronal cells, which sense nutrient availability, trigger feeding 

behavior. 13 

Intracerebroventricular (ICV) administration of glucose or long chain fatty 

acid has been found to inhibit food intake, 14 whereas central administration of 2deoxyglucose 

(2-DG), a non-metabolizable glucose analogue, or mercaptoacetate, 

an inhibitor of fatty acid oxidation, elicits feeding behavior. 15


Leptin, insulin, glucose, α-lipoic acid 

AMPK↓ 

Acetyl-CoA 

ACC↑ 

CPT-1↓ 

β-oxidation↓ 

Hypothalamus 

Malonyl-CoA↑ 

LCAC↑ 

Food intake↓ 

FIGURE 18.2 Possible mechanism by which decreased hypothalamic AMP-activated protein 

kinase (AMPK) activity reduces food intake. The reduction in hypothalamic AMPK 

activity in response to feeding-inhibiting factors such as leptin, insulin, glucose, and α- 

LA increases acetyl-CoA carboxylase (ACC) activity. Increased ACC activity leads to 

increase in malonyl-CoA levels, which in turn inhibits carnitine palmitoyltransferase-1 

(CPT-1) and mitochondrial β oxidation of long chain fatty acyl-CoA (LCAC). Recent 

studies suggest that increases in malonyl-CoA and/or LCAC levels in the hypothalamus 

decrease food intake. 16,18,28 

Several signaling pathways are thought to be involved in mediating nutrientinduced 

feeding. For example, central administration of the fatty acid synthase 

(FAS) inhibitors, cerulenin and C75, reduces food intake, but it can be prevented 

by the coadministration of the acetyl-CoA carboxylase (ACC) inhibitor, TOFA. 16 

This result suggests that malonyl-CoA, an intermediate metabolite between ACC 

and FAS, may be an anorexigenic signal (Figure 18.2). 

Patients with metabolic syndrome (MS) are characterized by insulin resistance, 

obesity, hyperlipidemia, premature atherosclerosis, and type-2 diabetes. It 

has been proposed that the common feature linking this syndrome is dysregulation 

of the AMPK/malonyl-CoA fuel-sensing and signaling network. 17 Both fuel surfeit 

and reduced AMPK activity result in increased cellular malonyl-CoA levels, 

which reduces fat oxidation and favors abnormal tissue accumulation of lipids. 

Some evidential factors indicate that activated AMPK and/or reduced malonyl- 

CoA levels may reverse these abnormalities or prevent them from occurring. In 

contrast, inhibition of hypothalamic carnitine palmitoyl transferase-1 (CPT-1) 

reduces food intake. 18 

These findings led to the suggestion that increased cytosolic concentrations 

of long chain fatty acyl-CoA and malonyl-CoA levels may serve as anorexigenic 

signals. Like pancreatic β cells, some neurons in the ventromedial and arcuate 

nuclei of the hypothalamus have glucose-sensing machinery that includes 

GLUT2, glucokinase, and the ATP-sensitive potassium (KATP) channel. 19 The


anorexic hormones leptin and insulin can activate the KATP channel in glucoseresponsive 

hypothalamic neurons. 20 

ANTIOBESITY FUNCTION THROUGH SUPPRESSION OF 

HYPOTHALAMIC AMPK 

REGULATION OF ENERGY EXPENDITURE AND FOOD INTAKE 

Chronic LA treatment significantly reduced body weight gain and visceral fat 

mass in obese Otsuta Long-Evans Tokushima fatty (OLETF) rats. 21 Male Sprague- 

Dawley rats given a standard chow containing LA for 2 weeks reduced food 

intake and body weight in a dose-dependent manner. Acute administration of LA 

by intraperitoneal injection (50, 75, or 100 mg/kg of body weight) also suppressed 

food intake. In addition, ICV injection of a small amount of LA reduced food 

intake, suggesting that the CNS is the primary site of the anorexic effect. 

Dietary administration of LA (0.5%, w/w) for 14 weeks also decreased body 

weight and visceral fat mass in genetically OLETF rats. This effect was accompanied 

by reductions in plasma glucose, insulin, free fatty acids, and leptin. It 

has been further demonstrated that LA improved endothelial dysfunction in obese 

rats by activating AMPK in endothelial cells by reducing glyceride and lipid 

peroxide and increasing NO synthesis. 22 

LA also increases whole body energy expenditure. One study compared body 

weight changes in rats given dietary LA (0.5%) and in pair-fed rats given the 

same amount of food. The LA-treated rats weighed significantly less than the 

pair-fed rats, indicating enhanced use of ingested energy. Energy expenditure 

measured by indirect calorimetry was higher in rats given LA than in control or 

pair-fed rats. UCP-1 in BAT is a chief regulator of energy expenditures in 

rodents. 23 UCP-1 is located in the mitochondrial inner membranes and dissipates 

proton electrochemical energy as heat. The pair-fed rats show reduced expression 

of UCP-1 in BAT and ectopic expression of UCP-1 in white adipose tissue. These 

results suggest that weight loss induced by LA is due in part to an enhancement 

of energy expenditure. 

The effects of LA on food intake and energy metabolism are similar to the 

reported effects of leptins 1 and 2. To determine whether the action of LA is 

mediated by leptin or leptin receptor signaling, leptin-deficient (Lep–/–) or leptin 

receptor-deficient (Lepr–/–) mice were fed a diet containing LA (0.5%). LA 

reduced food intake and caused weight losses in both strains of mice, indicating 

that leptin and its receptor are not essential for LA-induced anorexia. Leptin 

exerts its anorexic effect through the regulation of hypothalamic neuropeptides. 24 

Intraperitoneal (i.p.) administration of leptin (1 mg/kg) to Sprague-Dawley rats 

reduced the expression of hypothalamic neuropeptide Y (NPY) mRNA and 

increased that of pro-opiomelanocortin (Pomc) and corticotropin releasing hormone 

(Crh) mRNA. By contrast, the i.p. administration of LA (75 mg/kg) did 

not cause any acute changes in the levels of hypothalamic NPY, Pomc, Crh, promelanin 

concentrating hormone, or hypocretin mRNA.


ROLE OF HYPOTHALAMIC AMPK 

AMPK is an enzyme that acts as an intracellular energy sensor. 25 It is activated 

when cell energy status is low. When activated, AMPK inhibits ATP-consuming 

pathways (e.g., fatty acid synthesis) and activates ATP-generating pathways (e.g., 

fatty acid oxidation and glycolysis), thus maintaining energy balance within cells. 

AMPK activation in skeletal muscle enhances glucose uptake and mitochondrial 

fatty acid oxidation. 25 

In the liver, AMPK activation suppresses endogenous glucose production. 25 

In pancreatic β cells, AMPK seems to antagonize the effect of glucose on insulin 

secretion and induce β cell apoptosis. 26 Activation of AMPK has been reported 

to play a favorable role in preserving β cell function under lipid overloading 

conditions. 27 

Recent studies 21,28,29 have demonstrated that AMPK activity in hypothalamic 

neurons is altered by various factors and mediates their feeding effects. Hypothalamic 

AMPK activity is regulated by nutritional availability in hypothalamic 

neurons. Administration of 2-DG increases hypothalamic AMPK activity, while 

co-administration of an AMPK inhibitor, compound C, inhibited the 2-DGinduced 

glycolysis activity. 21 Conversely, ICV administration of glucose or restoration 

of food intake has been found to decrease hypothalamic AMPK activity. 29 

AMPK activity is reduced by ICV administration of anorexigenic hormones such 

as insulin and leptin, but increased by ICV administration of ghrelin, an orexigenic 

hormone. 28,29 In the hypothalamic paraventricular nucleus, AMPK activity is 

decreased by the MT-II melanocortin receptor agonist but increased by the melanocortin 

receptor antagonist, agouti-related protein (AgRP). 

Taken together, these findings indicate that AMPK is part of the common 

signaling pathway by which various factors regulate feeding behavior, that is, 

hypothalamic AMPK activity is reduced by feeding inhibiting factors and increased 

by feeding stimulating factors. Although the mechanism by which AMPK activity 

in hypothalamic neurons affects feeding behavior is still not fully understood, the 

leptin-induced reduction in hypothalamic AMPK activity is shown to decrease 

feeding by down-regulating expression of the NPY and AgRP orexigenic hormones. 

29 Alternatively, changes in AMPK activity may affect feeding via changes 

in intracellular malonyl-CoA concentrations and CPT-1 activity. 16,18 

REDUCTION OF FOOD INTAKE AND INCREASE OF ENERGY EXPENDITURE 

VIA SUPPRESSION OF HYPOTHALAMIC AMPK 

It was recently demonstrated that LA has anti-obesity effects mediated by the 

suppression of hypothalamic AMPK activity. 21 LA is an essential cofactor of 

mitochondrial pyruvate dehydrogenase and α-ketoacid dehydrogenase. In addition, 

LA enhances glucose metabolism in insulin-resistant rat skeletal muscle, 

and showed potent antioxidant activity by chelating transition metal ions and 

increasing cytosolic glutathione and vitamin C levels.


↓ Hypothalamic AMPK 

Food 

intake 

α−lipoic acid 

↑ UCP-1 in adipose tissue ↑ AMPK in muscle 

Energy 

expenditure Body weight 

FIGURE 18.3 Mechanism of body weight regulation by LA. LA reduces food intake and 

increases energy expenditure by suppressing hypothalamic AMP-activated protein kinase 

(AMPK) activity. It increases energy expenditure by increasing uncoupling protein (UCP)- 

1 expression in adipose tissue and by activating AMPK in skeletal muscle. 

Administration of LA to rodents reduces food intake and body weight as well 

as stimulating whole body energy expenditure. Central administration of very 

small amounts of LA increased UCP-1 mRNA in BATs, whereas co-administration 

of an AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside, 

prevented the LA-induced enhancement of energy expenditure and UCP-1 expression. 

21 These results indicate that food intake, energy expenditure, and UCP-1 

are mediated via hypothalamic AMPK inhibition by treatment with LA (Figure 

18.3). Finally, in contrast to its effects on the hypothalamus, LA increased glucose 

uptake and fatty acid oxidation in skeletal muscle by activating AMPK. 30 

The mechanism by which LA decreases hypothalamic AMPK activity is 

presently unknown. However, LA has been found to stimulate glucose transport 

and ATP synthesis in peripheral tissues, 31,32 and it may decrease hypothalamic 

AMPK activity by increasing glucose uptake and/or its metabolism in the hypothalamus. 

LA has been shown to activate ACC by decreasing ACC phosphorylation 

in hypothalamics. 21 Since activation of ACC can increase intracellular malonyl-CoA 

in hypothalamic neurons, malonyl-CoA may be a key downstream 

mediator of AMPK activity responsible for the decrease in food intake by LA 

administration as shown by Loftus et al. 16 and Ruderman and Prentki. 17


REGULATION OF ADIPOCYTE DIFFERENTIATION 

Obesity is closely correlated with the prevalence of diabetes and cardiovascular 

disease. Plasma levels of leptin, TNF-α, and non-esterified fatty acids are elevated 

in obesity and substantially contribute to the development of insulin resistance. 1 

Obesity is caused not only by hypertrophy of adipose tissue, but also by 

adipose tissue hyperplasia that triggers the transformation of pre-adipocytes into 

adipocytes. 9 

Adipocyte differentiation is a complex process that involves coordinated 

expression of specific genes and proteins associated with each stage of differentiation. 

This process is regulated by several signaling pathways. 33 Insulin, the 

major anabolic hormone, promotes in vivo accumulation of adipose tissue. 34 

Structurally unrelated inhibitors of PI3-K, LY294002 and wortmannin, have 

been shown to block adipocyte differentiation in a time- and dose-dependent 

fashion, 35 suggesting that the IR/Akt signaling pathway is important in transducing 

the pro-adipogenic effects of insulin. In contrast, mitogen-activated protein 

kinases (MAPKs) such as extracellular signal-regulated kinase (ERK) and c-Jun 

N-terminal kinase (JNK) suppress the process of adipocyte differentiation. 36,37 

TNF-α is known to exert its anti-adipogenic effects, at least in part, through 

activation of the ERK pathway. 36 However, p38K is shown to promote adipocyte 

differentiation. 38 

The signals that regulate adipogenesis either promote or block the cascades 

of transcription factors that coordinate the differentiation process. CCAAT element 

binding proteins (C/EBPs)-β and -δ and sterol response element binding 

protein-1 (ADD1/ SREBP1) are active during the early stages of the differentiation 

process and induce the expression and/or activity of peroxisome proliferatoractivated 

receptor-γ, (PPAR-γ), a pivotal coordinator of adipocyte differentiation. 

PPAR-γ, PPAR-α, and PPAR-δ are important regulators of lipid metabolism. 

Although they share significant structural similarities, the biological effects associated 

with each isotype are distinct. For example, PPAR-α and PPAR-δ regulate 

fatty acid metabolism, while PPAR-γ controls lipid storage and adipogenesis. 

Activated PPAR-γ induces exit from the cell cycle and, in cooperation with 

C/EBP-α, stimulates the expression of many metabolic genes such as Glut-4, 

lipoprotein lipase (LPL), 39 and adipocyte-specific fatty acid binding protein 

(aP2), 40 thus constituting a functional lipogenic adipocyte. 

JNK and ERK suppress this process by phosphorylating and thereby attenuating 

the transcriptional activity of PPAR-γ. 36,37 Besides these integral members 

of the adipogenesis program, other transcription factors such as AP-1 41 and cAMP 

responsive element binding protein (CREB) 42 are known to promote adipogenesis, 

whereas nuclear factor-κB (NF-κB) suppresses adipocyte differentiation. 43 

Therefore, the activity and/or expression of these transcription factors are 

attractive pharmacological targets for modulating adipocyte tissue formation and 

deposition.


INHIBITION OF ADIPOCYTE DIFFERENTIATION VIA MITOGEN- 

ACTIVATED PROTEIN KINASE PATHWAYS 

MAPK SIGNALING PATHWAYS MEDIATE ACTIONS OF LA ON 

ADIPOGENESIS 

Several lines of evidence indicate that pro-adipogenic transcription factors such 

as PPAR-γ and members of the C/EBP family can be negatively regulated by 

MAPKs. Epidermal growth factor, platelet-derived growth factor, lipoxygenase- 

1 metabolites, and prostaglandin F 2α phosphorylate and attenuate transcriptional 

activity of PPAR-γ by activating MAPK signaling pathways. 44–46 Similarly, LA 

treatment of pre-adipocytes inhibits the insulin- or hormonal cocktail-induced 

transcriptional activity of PPAR-γ and C/EBPα, which is accompanied by strong 

activation of ERK and JNK. 

Inhibitors of ERK and JNK activity abolish the inhibitory effect of LA on 

insulin- or hormonal cocktail-induced adipogenesis. On the other hand, LA hardly 

stimulates phosphorylation of insulin receptor (IR) or insulin receptor substrate 

(IRS)-1 in pre-adipocytes and adipocytes at the early stages of differentiation. In 

particular, upon LA treatment, a transient Akt phosphorylation is detected in preadipocytes 

although it is not detectable in adipocytes at early stages of differentiation. 

In contrast, insulin strongly activates IR and IRS-1 and induces longlasting 

Akt activation in pre-adipocytes and in adipocytes at early stages of 

differentiation. These findings exclude possible involvement of Akt activation in 

LA-induced inhibition of adipogenesis and demonstrate that LA down-regulates 

PPAR-γ and C/EBP-α through activation of MAPK signaling pathways such as 

ERK and JNK (Figure 18.4). 

MODULATION OF AUXILIARY TRANSCRIPTION FACTORS IN ADIPOGENESIS 

Transcriptional activities of AP-1 and CREB are increased in fully differentiated 

3T3-L1 adipocytes as well as after 2-hr treatment with a hormonal cocktail in 

3T3-L1 pre-adipocytes. AP-1 is involved in transcriptional regulation of aP2 and 

LPL genes. 47,48 CREB appears to stimulate transcription of several adipocytespecific 

genes such as aP2, fatty acid synthetase, and phosphoenolpyruvate carboxykinase. 

42 LA, however, strongly down-regulates AP-1 and CREB activities, 

whereas it up-regulates NF-κB activities in pre-adipocytes. 

Many anti-adipogenic factors such as pro-inflammatory cytokines, 49 TNF-α, 50 

and endrin 51 are also known to up-regulate NF-κB activity, whereas pro-adipogenic 

factors such as troglitazone display opposite effects in 3T3-L1 cells. 52 

Considering that AP-1, 53 CREB, 54 and NF-κB 55 mediate major downstream effects 

of MAPK signaling pathways, our findings suggest that activation of the MAPK 

signaling pathways by LA leads to differential regulation of these transcription 

factors, which eventually results in decreased expression of the adipocyte-specific 

genes, and consequently suppresses adipogenesis.


Insulin 

IR/PI3-K/Akt 

Early gene expression 

(c-Myc, c-Fos, c-Jun) 

Clonal expansion 

FIGURE 18.4 Regulation of adipocyte differentiation by LA. LA activates both the 

IR/PI3-K/Akt pathway and the MAPK pathway. Activation of the MAPK pathway mainly 

leads to the inhibition of pro-adipogenic transcription factors such as PPAR-γ, C/EBP-α, 

AP-1, and CREB, and the activation of anti-adipogenic factor such as NF-κB. The inhibition 

of pro-adipogenic transcription factors decreases transcription of several proteins 

including LPL and aP2, which are two major markers of adipogenesis. In contrast, activation 

of NF-κB may enhance cytokine release and inhibit adipogenesis. LA may also 

prevent the adipogenic process by regulating the expression of immediate early genes, 

which are involved in the process of clonal expansion. 

MAPK SIGNALING PATHWAYS MEDIATE LA ACTIONS ON CELL CYCLE 

AND CLONAL EXPANSION 

AP-1 

CREB 

α-Lipoic acid 

LPL, aP2 

MAPK 

PPAR-γ 

C/EBP-α 

NF-κB 

Cytokines 

(TNF-α, IL-6) 

Preadipocytes 

Anti-adipogenesis 

Dedifferentiation 

Adipocytes 

In the course of adipogenesis, one of the first events following hormonal induction 

is re-entry of growth-arrested pre-adipocytes into the cell cycle. LA has been 

demonstrated to inhibit the process of clonal expansion when induced by insulin 

or a hormonal cocktail, indicating that insulin and LA oppositely regulate cell 

cycle progression. This differential effect seems to be due to the potency and/or 

the kinetics of activating of MAPK and IR/Akt signaling pathways. 

Both insulin and LA activate MAPK signaling pathways in pre-adipocytes. 

However, insulin, but not LA, also strongly activates the IR/Akt signaling pathway. 

This observation indicates that progression in the cell cycle and clonal 

expansion may require activation of both MAPK and IR/Akt signaling pathways. 

On the other hand, insulin-induced MAPK activation is transient, whereas that 

of LA lasts longer, indicating that duration of MAPK activation may be another 

important factor in determining the fate of a cell in the cell cycle. Indeed, transient 

activation of MAPK has been considered as a contributor to cell cycle progression 

whereas its prolonged activation can result in cell cycle arrest via induction of 

p21 Cip1/Waf1 expression and inhibition of cyclin-dependent kinase activity. 56,57


It should be emphasized that JNK is known to activate p53, which triggers 

activation of several proteins involved in cell cycle arrest such as p21 Cip1/Waf1 . 58 

This evidence supports the notion that activation of MAPKs mediates the inhibitory 

effect of LA on the clonal expansion process by suppressing the expression 

of immediate early genes. 

SUMMARY 

AMPK is an enzyme that functions as an intracellular energy sensor. It is activated 

when the energy status of a cell is low. LA decreases hypothalamic AMPK activity 

and results in profound weight losses in rodents by reducing food intake and enhancing 

energy expenditures. However, LA increases AMPK activity in skeletal muscle 

and other organs, which enhances glucose uptake and mitochondrial fatty acid oxidation. 

Activation of hypothalamic AMPK activity reverses by LA administration, 

which in turn reduces food intake and increases energy expenditure. ICV administration 

of glucose decreases hypothalamic AMPK activity, whereas inhibition of 

intracellular glucose utilization through the administration of 2-DG increases hypothalamic 

AMPK activity and food intake. 2-DG-induced hyperphagia is reversed by 

inhibiting hypothalamic AMPK. These findings indicate that hypothalamic AMPK 

is important in the regulation of food intake and energy expenditure and that LA 

exerts anti-obesity effects by suppressing hypothalamic AMPK activity. 

It was reported that LA inhibited differentiation of pro-adipocytes induced 

by a hormone mixture or troglitazone. Northern blot analysis of cells demonstrated 

that this inhibition was accompanied by attenuated expression of aP2 and 

LPL. Electrophoretic mobility shift assay and western blot analysis of cells 

demonstrated that LA modulates transcriptional activity and/or expression of antior 

pro-adipogenic transcriptional factors. 

LA treatment of pre-adipocytes also resulted in prolonged activation of major 

MAPK signaling pathways. These findings suggest that LA inhibits insulin- or 

hormonal mixture-induced differentiation of pre-adipocytes by modulating activity 

and/or expression of pro- or anti-adipogenic transcriptional factors mainly by 

activating the MAPK pathways. 

In conclusion, LA may be beneficial in obesity via two major mechanisms. 

It may regulate food intake and energy expenditure by decreasing hypothalamic 

AMPK activity and block adipocyte differentiation by activating MAPK pathways. 

Further studies are warranted to evaluate food intake and energy expenditure 

via hypothalamic AMPK activity and adipocyte differentiation by MAPK 

pathways in humans. 


We thank Drs. Ki-Up Lee and Jong-Yeol Park of the University of Ulsan College 

of Medicine, Asan Medical Center, Seoul, Republic of Korea, for providing their 

data. This study was supported by Brain Korea 21 Grant M103300.


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281, 809.

19 

CONTENTS 

Effects of Conjugated 

Linoleic Acid and Lipoic 

Acid on Insulin Action in 

Insulin-Resistant Obese 

Zucker Rats 


Abstract .............................................................................................................289 

Introduction .......................................................................................................290 

Regulation of Skeletal Muscle Glucose Transport System..............................291 

Etiology of Insulin Resistance in Obese Zucker Rats .....................................292 

Nutriceutical Interventions: Conjugated Linoleic Acid and α-Lipoic Acid ....292 

Effects of CLA in Obese Zucker Rats....................................................292 

Effects of ALA in Obese Zucker Rats....................................................293 

Interactions of CLA and ALA in Obese Zucker Rats............................294 

Perspectives and Future Directions ..................................................................295 

References .........................................................................................................296 

ABSTRACT 

Insulin-resistant conditions such as pre-diabetes and type 2 diabetes are characterized 

by defects in the ability of insulin to activate glucose transport in 

skeletal muscle. One animal model that has proven useful in elucidating the 

multifactorial etiology of skeletal muscle insulin resistance is the obese Zucker 

(fa/fa) rat, characterized by complete leptin resistance, massive central obesity, 

hyperinsulinemia, dyslipidemia, and oxidative stress (the imbalance between 

exposure of tissue to an oxidant stress and cellular antioxidant defenses). 

Studies published by our research group addressed the utility of two nutriceutical 

compounds, conjugated linoleic acid (CLA) and alpha-lipoic acid (ALA), 

both of which possess antioxidant properties, in improving the metabolic 

289


conditions of obese Zucker rats. These studies indicate that chronic administration 

of the R-enantiomer of ALA (R-ALA) to obese Zucker rats improved 

whole-body insulin sensitivity and enhanced the insulin-stimulated skeletal 

muscle glucose transport system, at least in part by up-regulation of IRS-1dependent 

insulin signaling. CLA treatment of obese Zucker rats improved 

glucose tolerance and insulin-stimulated glucose transport activity, attributable 

exclusively to the trans-10, cis-12 isomer and associated with reductions in 

oxidative stress and muscle lipid levels. Significant interactions exist between 

CLA and R-ALA for enhancement of insulin action on skeletal muscle glucose 

transport in the obese Zucker rat, also ascribed to reductions in muscle oxidative 

stress and lipid storage. Collectively, these investigations support the 

fundamental concept that oxidative stress is an important component of the 

etiology of insulin resistance that can be beneficially modulated by antioxidant 

interventions, including CLA and ALA. 

INTRODUCTION 

Insulin resistance of skeletal muscle glucose disposal is a primary defect associated 

with the development of the pre-diabetic state and with the conversion from 

a pre-diabetic state to overt type 2 diabetes. 1,2 Pre-diabetes is defined as a condition 

in which fasting blood glucose levels are elevated above normal (100 to 

125 mg/dl) but do not exceed the threshold for the diagnosis of diabetes (>126 

mg/dl). 3 Pre-diabetes is frequently characterized by several other cardiovascular 

risk factors including visceral obesity, hyperinsulinemia, and dyslipidemia, 

encompassing a condition of elevated cardiovascular disease risk known as metabolic 

syndrome (see Chapter 1). It is estimated over 40 million individuals in 

the United States have pre-diabetes. 3 In the face of further metabolic destabilization 

(primarily via ß cell dysfunction), this population could increase substantially 

the number of overt type 2 diabetics (presently estimated to be >20 million) 

in the U.S. While the etiology of the skeletal muscle insulin resistance present 

in pre-diabetic and type 2 diabetic individuals is clearly multifactorial, accumulating 

evidence indicates that one factor that can cause insulin resistance and 

exacerbate existing insulin resistance is oxidative stress, the imbalance between 

exposure of tissues to oxidant stresses and cellular antioxidant defenses. 4 

A major challenge to the scientific community and to healthcare professionals 

is the design and implementation of effective interventions to reduce insulin 

resistance in these pre-diabetic and type 2 diabetic populations. Increased physical 

activity and loss of visceral fat have long been the preferred interventions for 

ameliorating insulin resistance, 5 but these lifestyle changes are difficult to implement 

and maintain in human pre-diabetic and type 2 diabetic subjects. Therefore, 

pharmaceutical and nutriceutical treatments are seen as viable alternative therapies 

for reducing insulin resistance in these individuals. 

Two nutriceutical compounds that received considerable attention recently 

as interventions in insulin-resistant states are conjugated linoleic acid (CLA)

Effects of Conjugated Linoleic Acid and Lipoic Acid 291 

and alpha-lipoic acid (ALA). 6,7 Both substances have antioxidant properties, 

and their utility in the context of oxidative stress-associated insulin resistance 

of skeletal muscle glucose disposal will be the focus of this chapter. The normal 

regulation of the glucose transport system in skeletal muscle will be described 

first, followed by a brief section summarizing the fundamental defects of the 

glucose transport system, including the contribution of oxidative stress, that 

lead to the insulin-resistant state and ultimately to the development of overt 

type 2 diabetes. Finally, a discussion of potential physiological and pharmacological 

interventions in the prevention and treatment of insulin resistance is 

included, culminating with more detailed coverage of how CLA and ALA, 

individually and in combination, can modify oxidative stress and reduce insulin 

resistance of skeletal muscle glucose transport in the pre-diabetic state. This 

discussion will focus on the obese Zucker (fa/fa) rat, an animal model that 

displays complete leptin resistance, massive visceral obesity, glucose intolerance, 

insulin resistance, hyperinsulinemia, dyslipidemia, and oxidative stress, 

and clearly reflects many of the pathophysiological conditions present in 

humans with pre-diabetes and metabolic syndrome. 

REGULATION OF SKELETAL MUSCLE GLUCOSE 

TRANSPORT SYSTEM 

The acute regulation of the insulin-dependent glucose transport system in mammalian 

skeletal muscle occurs via the sequential activation of several intracellular 

proteins. 8,9 This cascade is initiated by insulin binding to the α subunit of the 

insulin receptor (IR), thereby activating the intrinsic tyrosine kinase of the transmembrane 

IR ß subunits. The activated IR tyrosine kinase can phosphorylate a 

number of important intracellular substrates, including isoforms of insulin receptor 

substrate (IRS1-4). The most important isoforms in skeletal muscle are primarily 

IRS-1 and IRS-2. Tyrosine-phosphorylated IRS-1 or IRS-2 can then interact 

with the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3-kinase), 

increasing the catalytic activity of the p110 subunit of PI3-kinase. Phosphoinositide 

moieties produced by PI3-kinase subsequently activate 3-phosphoinositidedependent 

kinases (PDKs) that then phosphorylate and activate Akt, a serine/threonine 

kinase, and atypical protein kinase C isoforms. The activation of these steps 

eventually results in the translocation of the glucose transporter protein isoform 

GLUT-4 to the sarcolemmal membrane where glucose transport takes place via 

a facilitative diffusion process. 

Glucose transport in skeletal muscle can also be activated by contractile 

activity. 10 The intracellular mechanisms responsible for this insulin-independent 

stimulation of glucose transport include the activation of 5-adenosine-monophosphate-activated 

protein kinase (AMP kinase), an enzyme stimulated by a decrease 

in cellular energy charge, 11–14 and activation of calcium and calmodulin-dependent 

protein kinase. 14,15 Ultimately, these contraction-associated signaling factors stimulate 

GLUT-4 translocation 16,17 and enhanced glucose transport activity.


ETIOLOGY OF INSULIN RESISTANCE IN OBESE 

ZUCKER RATS 

The obese Zucker (fa/fa) rat develops numerous pathophysiological conditions 

because of a mutation in the leptin receptor gene. 18–20 This defect results in chronic 

hyperphagia and eventually in obesity, especially extensive visceral obesity. Skeletal 

muscle insulin resistance in obese Zucker rats is reflected in diminished 

insulin-stimulated GLUT-4 protein translocation 21,22 and glucose transport activity. 

22–24 The compromised glucose transport system in muscles of obese Zucker 

rats likely results from specific defects in the insulin signaling cascade including 

reduced IRS-1 protein expression 25–27 and diminished insulin-stimulated IRS-1 

tyrosine phosphorylation and PI3-kinase activation. 25,27 Moreover, the obese 

Zucker rat is markedly dyslipidemic and displays reduced activity of beneficial 

protein kinase C isoforms 28 and overactivation of deleterious protein kinase C 

isoforms 29,30 that may be important in the etiology of insulin resistance in this 

pre-diabetic animal model. 

This abnormal functionality of the insulin signaling cascade in skeletal muscle 

is also characteristic of humans with pre-diabetes and overt type 2 diabetes. 

Defects in human insulin-resistant skeletal muscle include impaired insulin stimulation 

of tyrosine phosphorylation of IR and IRS-1 and of the activities of PI3kinase 

and Akt 31–34 and reduced GLUT-4 protein translocation. 35,36 

The obese Zucker rat also displays characteristics of oxidative stress that may 

be associated with its well established insulin-resistant state. For example, intramuscular 

triglycerides and protein carbonyls — markers of oxidative damage 37 

— are elevated in the skeletal muscles, cardiac muscles, and livers of these 

animals 38–40 and are associated with diminutions of insulin action on the IR/IRS- 

1-dependent signaling pathway. 27 We have also demonstrated that in vitro oxidant 

stress (~90 µM H 2O 2) can directly impair insulin action on IR/IRS-1-dependent 

signaling, glucose transport, and glycogen synthesis in otherwise insulin-sensitive 

skeletal muscles from lean Zucker rats (Kim, J.S., Saengsirisuwan, V., and Henriksen, 

E.J., manuscript submitted), providing additional support for the concept 

that oxidative stress can play a role in the etiology of the insulin-resistant state 

in skeletal muscle. 

NUTRICEUTICAL INTERVENTIONS: CONJUGATED 

LINOLEIC ACID AND α-LIPOIC ACID 

EFFECTS OF CLA IN OBESE ZUCKER RATS 

CLA is a dienoic derivative of linoleic acid that exists primarily as one of two 

isomers — the cis-10, trans-12 isomer and the trans-10, cis-12 isomer, with 

numerous other minor isoforms possible. CLA possesses significant antioxidant 

properties 41 and has been used as an intervention against cancer and heart disease. 

42 Chronic CLA administration diminished visceral fat in obese Zucker rats 39 

and adult humans. 43 In the Zucker diabetic fatty (ZDF) rat, a model of overt type


2 diabetes, the trans-10, cis-12 isomer of CLA enhanced glucose tolerance, 

reduced fasting plasma glucose, insulin, and free fatty acids, and increased insulin-stimulated 

skeletal muscle glucose transport activity and glycogen synthase 

activity. 44,45 This supports its potential as an intervention for treatment of type 2 

diabetes, possibly as an activator of the peroxisome proliferator-activated receptor-γ 

in a variety of tissues. 44 

Our research group conducted investigations of the metabolic actions of CLA 

in pre-diabetic obese Zucker rats. In this model of pre-diabetes, as in diabetic 

ZDF rats, 44,45 CLA treatment led to enhanced glucose tolerance, increased wholebody 

insulin sensitivity, decreased fasting plasma glucose, insulin, and free fatty 

acid levels, and improved insulin-mediated skeletal muscle glucose transport 

activity. 39,40 Importantly, CLA treatment caused a significant decrease in visceral 

fat in this grossly obese animal model. 39 

These metabolic actions of CLA can be attributed solely to the trans-10, cis- 

12 isomer, as the cis-10, trans-12 isomer is metabolically neutral in this animal 

model. Moreover, these trans-10, cis-12-CLA-induced metabolic improvements 

in the obese Zucker rat are highly correlated with reductions in skeletal muscle 

protein carbonyl levels and intramuscular triglyceride levels. 39 This latter finding 

is of importance in light of the well-recognized role of elevated muscle lipid in 

the etiology of insulin resistance. 46 Collectively, these results highlight the potential 

of the trans-10, cis-12 isomer of CLA as a nutriceutical intervention in specific 

conditions of glucose intolerance and insulin resistance, and indicate that the 

beneficial metabolic actions of CLA may be related to its ability to reduce 

intramuscular lipid levels and local indices of oxidative stress. 

EFFECTS OF ALA IN OBESE ZUCKER RATS 

The metabolic actions of ALA in pre-diabetic obese Zucker rats have been 

extensively investigated by our research group in the last decade, and most of 

these findings are summarized in recent review articles and chapters. 4,6,47 The 

highlights of these studies will be briefly covered in this section. 

The vast majority of the beneficial metabolic actions of ALA in obese Zucker 

rats can be attributed to the R-enantiomer, with the S-enantiomer being either 

metabolically neutral or even eliciting some metabolic worsening such as 

decreased muscle GLUT4 glucose transporter protein expression. 48 R-ALA 

acutely enhances glucose transport and intracellular glucose metabolism in both 

insulin-sensitive 49,50 and insulin-resistant 48,50,51 skeletal muscle preparations. 

Chronic administration (2 to 6 weeks) of R-ALA to obese Zucker rats resulted 

in improvements of whole-body glucose tolerance and insulin sensitivity, reductions 

in fasting plasma insulin and lipids, and enhanced insulin-stimulated glucose 

transport activity in isolated skeletal muscle. 27,38 

The increased insulin action on skeletal muscle glucose transport associated 

with chronic R-ALA treatment of obese Zucker rats is correlated with a proportional 

reduction of oxidative stress, as reflected by muscle protein carbonyl 

levels. 38 This antioxidant intervention is associated with reductions in cardiac and


liver protein carbonyl levels. 38 Chronic treatment of obese Zucker rats with R- 

ALA also induced dose-dependent reductions in plasma free fatty acids that 

correlated with improvements in whole-body glucose tolerance and insulin sensitivity 

and with improved insulin action on the skeletal muscle glucose transport 

system. 38,52 It now appears that R-ALA treatment can improve functionality of 

the IRS-1-dependent insulin signaling pathway in skeletal muscles of obese 

Zucker rats. Chronic administration of R-ALA to this insulin-resistant, pre-diabetic 

animal model induces an enhancement of IRS-1 protein expression and 

increased insulin-stimulated IRS-1 associated with the p85 subunit of phosphatidylinositol-3-kinase, 

a surrogate measure of PI3-kinase activation. 27 

These results on the beneficial effects of chronic treatment with ALA in obese 

Zucker rats were confirmed recently in another rodent model of obesity-associated 

insulin resistance and glucose dysregulation, the Otsuka Long-Evans Tokushima 

Fatty (OLETF) rat. 53 OLETF rats treated with ALA displayed reduced body 

weights, decreased blood glucose levels, and lower intramuscular triglyceride 

concentrations. 53 These investigators made the novel finding that the reduction in 

muscle lipids and the increase in muscle glucose uptake in short-term ALA-treated 

OLETF rats may be associated with the activation of AMP kinase. 54 

Several clinical investigations generally supported the animal model-based 

findings on the effects of ALA on glucoregulation. The acute infusion of ALA 

in type 2 diabetic human subjects resulted in ~30% improvement in glucose 

disposal rate during a euglycemic, hyperinsulinemic clamp. 55,56 Moreover, the 

chronic intravenous or oral administration of ALA to type 2 diabetic subjects 

also elicited significant improvements in whole-body insulin sensitivity. 57–59 The 

results from this limited number of clinical investigations, combined with the 

findings from animal models, provide further support for additional clinical studies 

on the effectiveness of ALA in the treatment of insulin resistance in prediabetic 

and type 2 diabetic humans. 

INTERACTIONS OF CLA AND ALA IN OBESE ZUCKER RATS 

To our knowledge, there is only one published investigation related to potential 

interactive effects of CLA and ALA to modulate insulin action in insulin-resistant 

skeletal muscle. The study, performed by our research group, was published in 

2003. 40 Obese Zucker rats were treated chronically with either a mixture of CLA 

isomers, with R-ALA, or with a combination of R-ALA and CLA, at low, 

minimally effective doses, or at high, maximally effective doses. 

While the high doses of CLA and R-ALA individually induced significant 

metabolic improvements in these pre-diabetic animals, including increased insulin-stimulated 

glucose transport activity and reduced muscle oxidative stress and 

lipid levels in skeletal muscle, the combination of these high doses of CLA and 

R-ALA did not provide for any further metabolic enhancements above those 

brought about by the individual agents. In contrast, the combination of low doses 

of CLA and R-ALA (which individually produced minimal metabolic actions) 

resulted in substantial improvements in glucose tolerance and whole-body insulin


sensitivity and caused significant increases in insulin-stimulated glucose transport 

activity in skeletal muscle that were closely associated with reductions in muscle 

oxidative stress and lipid levels. This novel synergistic interaction between the 

fatty acid CLA and the metabolic antioxidant R-ALA supports the use of combined 

CLA and R-ALA in the modulation of whole-body and skeletal muscle 

insulin resistance. 

PERSPECTIVES AND FUTURE DIRECTIONS 

The information discussed in this chapter supports the beneficial role of the R- 

ALA and CLA nutriceutical compounds in the modulation of insulin action in 

insulin-resistant, pre-diabetic obese Zucker rats. With regard to these effects of 

R-ALA, the general consensus is that this antioxidant and reactive sulfhydrylcontaining 

agent elicits improvements in glucose tolerance, insulin sensitivity, 

IR/IRS-1-dependent insulin signaling, and muscle glucose transport capacity. 

While some variability in the absolute effect of ALA administration on these 

variables in both animal models and in human type 2 diabetics does exist in the 

literature, this can generally be attributed to differences among studies in the 

ALA dose given, the route of ALA administration, the release rate of the ALA, 

and the initial degree of insulin resistance present in the experimental subjects. 

On the other hand, several investigations utilizing both animal models and 

human subjects presented contradictory results arising from chronic CLA administration. 

In some investigations, CLA administration actually worsened insulin 

sensitivity in obese mice 60,61 and in abdominally obese men. 62,63 At least part of 

this controversy can be accounted for by differential responses to the CLA in the 

inherently different animal models employed. For example, the chronic administration 

of trans-10, cis-12-CLA to obese Zucker rats 39 or ZDF rats 45 caused a 

relatively small decrease in fat mass and an increase in whole-body and skeletal 

muscle insulin sensitivity, whereas this same CLA administration to obese 

C57BL/6 mice elicited a relatively large fat mass loss but induced insulin resistance. 

60,61 The fundamentally different metabolic response to CLA in the mouse 

model can likely be ascribed to the development of a state of lipodystrophy 

marked by an extreme fat mass deficit and impaired insulin action. In contrast, 

the CLA-treated obese Zucker and ZDF rats retained a morbidly obese phenotype. 

The specific animal model employed and human population under investigation 

must be taken into consideration when assessing the potential for CLA to bring 

about beneficial modulation of metabolic parameters. 

Finally, based on the results of our study in obese Zucker rats, 40 an important 

area for future clinical investigation would be the assessment of the effects of 

combined low doses of CLA and R-ALA on metabolic parameters in obese 

humans with insulin resistance, glucose intolerance, and dyslipidemia. There are 

novel interactions of these two nutriceutical compounds that may be exploited in 

the treatment of human insulin-resistant states, especially those states associated 

with visceral adiposity.


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20 

CONTENTS 

Trivalent Chromium 

Supplementation 

Inhibits Oxidative Stress, 

Protein Glycosylation, 

and Vascular 

Inflammation in High 

Glucose-Exposed 

Human Erythrocytes and 

Monocytes 


Abstract .............................................................................................................301 

Introduction .......................................................................................................302 

Pro-Inflammatory Cytokines and Vascular Inflammation in Diabetes.............302 

Oxidative Stress and Vascular Inflammation....................................................303 

Trivalent Chromium and Diabetes....................................................................303 

Conclusion.........................................................................................................307 


References .........................................................................................................308 

ABSTRACT 

Epidemiological studies have shown lower levels of chromium among men with 

diabetes and cardiovascular disease (CVD) compared with healthy control 

301


subjects. The mechanism by which chromium may decrease the incidence of 

CVD and insulin resistance is not known. This study demonstrates that chromium 

inhibits glycosylation of proteins, oxidative stress, and pro-inflammatory cytokine 

secretion, all of which are risk factors in the development of CVD. Erythrocytes 

or monocytes isolated from fresh human blood were treated with high concentrations 

of glucose (mimicking diabetes) in the presence or absence of chromium 

chloride in a medium at 37˚C for 24 hr. We observed that chromium supplementation 

prevented increases in protein glycosylation and oxidative stress caused by 

high levels of glucose in erythrocytes. In monocytes, chromium supplementation 

inhibited the high glucose-induced secretion of interleukin (IL)-6 and tumor 

necrosis factor (TNF)-α. This study demonstrates that chromium supplementation 

protects against oxidative stress and vascular inflammation caused by exposure 

to high glucose in blood cells and provides evidence for a novel mechanism by 

which chromium supplementation may decrease the incidence of CVD in diabetic 

patients. 

INTRODUCTION 

Vascular inflammation and CVD are the leading causes of morbidity and mortality 

in the diabetic population and remain major public health issues. Hyperglycemia 

is one of the major risk factors in the development of vascular complications. 1 

Intensive blood glucose control dramatically reduces the devastating complications 

that result from poorly controlled diabetes. Diabetes is treated with diet and 

insulin administration. However, for many patients, achieving tight glucose control 

is difficult with current regimens. The risk of CVD in diabetics is three- to 

five-fold greater than that of the general population. 

PRO-INFLAMMATORY CYTOKINES AND VASCULAR 

INFLAMMATION IN DIABETES 

TNF-α and IL-6 are pro-inflammatory cytokines produced by macrophages and 

other cell types in response to various stimuli. 2,3 The levels of these cytokines 

are elevated in the blood of many diabetic patients. 4–10 Increases in circulating 

levels of TNF-α and IL-6 decrease insulin sensitivity, 2,10–16 which can necessitate 

higher doses of insulin to maintain glycemic control in type 1 diabetic patients. 

Increased levels of insulin administration carry a risk of hypoglycemia. In addition, 

elevated circulating levels of TNF-α and IL-6 can induce expression of 

adhesion molecules and thus monocyte–endothelial cell adhesion, now recognized 

as an early and rate-limiting step in vascular inflammation and the development 

of vascular disease. 17–23 

Different human endothelial cells may have distinct ICAM forms and expression 

mechanisms. 24 Expression of VCAM-1 independent of systemic levels of 

TNF-α has been shown in pulmonary endothelial cells. 17 Several genes associated 

with adhesion molecules (ICAM-1, VCAM-1) and inflammatory cytokines are

Trivalent Chromium Supplementation 303 

regulated by NFκB. 25,26 The agents that suppress NFκB activation can diminish 

cell adhesion and vascular inflammation. 

OXIDATIVE STRESS AND VASCULAR INFLAMMATION 

Oxidative stress can also influence the expression of multiple genes in vascular 

cells, including signaling molecules such as PKC, NFκB, and ERK. 27–32 Overexpression 

of these genes may lead to endothelial dysfunction and ultimately to 

microvascular and macrovascular disease. High glucose (HG) can up-regulate 

expression of transcription factors, such as NFκB, activating protein-1 and the TNFα 

genes in monocytes. 31 TNF-α accumulation in conditioned media increased 10fold 

and mRNA levels were increased 11.5-fold by HG. 31 This indicates that both 

NFκB and AP-1 mediated enhanced TNF-α transcription by HG. 31 

This expression of the TNF-α gene is mediated by the protein kinases p38 

and JNK-1, which are respectively dependent on and independent of oxidative 

stress pathways. 30,31 Several studies advocate the importance of the p38 pathway 

in diabetes. 30 cAMP-dependent protein kinases (PKAs) can activate phosphorylation 

of substrate proteins and cross-talk with MAPK pathways and proteins 

involved in signal transduction pathways, leading to altered gene expression and 

modulation of physiological processes. 32–37 cAMP is known to modulate cytokine 

production in a number of cell types. 32–37 This suggests that oxidative stress plays 

a key role in the regulatory pathway that progresses from elevated glucose to 

monocyte and endothelial cell activation in the enhanced vascular inflammation 

of diabetes. 21,38 

TRIVALENT CHROMIUM AND DIABETES 

Trivalent chromium, the reduced form of the element, is required for insulin 

action. 39–44 A chromium-containing oligopeptide present in insulin-sensitive cells 

binds to the insulin receptor, markedly increasing the activity of the insulinstimulated 

tyrosine kinase. 39,40 Overt chromium deficiency has been demonstrated 

in patients receiving total parenteral nutrition without chromium supplementation. 

45 It is characterized by hyperglycemia, glycosuria, and weight loss that 

cannot be controlled with insulin. 45,46 As a consequence, total parenteral nutrition 

solutions are regularly supplemented with chromium. 47 Intraperitoneal injections 

of potassium chromate also reversed atherosclerotic plaques in rabbits. 48,49 

The main route of exposure to chromium in the general population is dietary 

intake. Most chromium in the diet is trivalent, and any hexavalent chromium in 

food or water is reduced to the trivalent form in the acidic environment of the 

stomach. 50,51 Foods with high chromium concentrations include whole grain products, 

green beans, broccoli, and bran cereals. 52 The chromium contents of meats, 

poultry, and fish vary widely because chromium may be introduced during transport, 

processing, and fortification. 52 Foods rich in refined sugars are low in 

chromium and actually promote chromium loss. 53


Based on the chromium contents of well balanced diets, 52 adequate intake 

values in adults have been established at 35 μg/day for men and 25 μg/day for 

women. 51 Although there are no national survey data covering chromium intake, 51 

a study of self-selected diets of U.S. adults indicated that the chromium intakes 

of a substantial proportion of subjects may be well below the adequate intake. 54 

Similar results have been shown in the United Kingdom, Finland, Canada, and 

New Zealand. 55 Thus, subclinical chromium deficiency may be a contributor to 

glucose intolerance, insulin resistance, and cardiovascular disease, particularly in 

aging populations and populations that have increased chromium requirements 

because of high sugar diets. 43 

Concentrations of chromium in the blood, lenses, and toenails are lower in 

diabetic patients compared with concentrations in the normal population. 46,56,57 

Several studies have suggested that chromium supplementation may be beneficial 

in individuals with type 2 and type 1 diabetes, gestational diabetes, or steroidinduced 

diabetes, as evidenced by decreased blood glucose values or decreased 

insulin requirements. 42,43,58–67 Although the majority of research on diabetes has 

focused on type 2, a few small studies have tested the efficacy of Cr 3+ on type 1 

diabetes and found it effective. 64 One study supplemented 162 patients (48 with 

type 1 diabetes, the remainder with type 2) with 200 μg/day Cr 3+ picolinate. 

Seventy-one percent of the type 1 patients responded positively, allowing 30% 

decreases in insulin doses. Blood sugar fluctuations also responded positively, 

decreasing as early as 10 days after treatment. Supplementation of chromium as 

the picolinate (600 μg/day) in a 28-year-old woman with an 18-year history of 

type 1 diabetes reduced HbA 1C from 11.3 to 7.9% in 3 months. 65 When patients 

receiving total parenteral therapy were supplemented with chromium, their diabetes 

symptoms reversed and they required smaller doses of exogenous insulin. 66 

In atherosclerotic rabbits, an injection of chromium chloride resulted in 

marked reductions in the plaques covering the aortic intimal surfaces, aortic 

weights, and cholesterol contents. 49 Chromium can reduce elevated cholesterol 

and triglycerides in a dose-dependent relationship. 68 Results from two casecontrol 

studies suggest an inverse association between chromium levels in toenails 

and risks of myocardial infarction in the general population. 69 Similarly, a recent 

report of the Health Professionals Follow-up Study found lower levels of toenail 

chromium among men with diabetes and CVD compared with healthy control 

subjects. 56 

However, randomized trials of chromium supplementation in diabetes have 

not been definitive. Many studies have not been blinded, used inappropriate 

glucose metabolism assessment parameters, or included heterogeneous and 

poorly characterized patient populations. More rigorous blinded and well controlled 

studies are needed to fully assess the efficacy and mechanism of the action 

of Cr 3+ supplementation as an adjuvant therapy for diabetic patients. 

Figure 20.1 illustrates the effects of high glucose (HG) and trivalent 

chromium on TNF-α secretion in peripheral blood mononuclear cells (PBMCs) 

isolated from normal human blood. HG resulted in a significant increase in 

TNF-α secretion in PBMCs. Supplementation with chromium caused a


TNF-α Secretion (pg/ml) 

2700 

2500 

2300 

2100 

1900 

1700 

1500 

* 

# 

** 

FIGURE 20.1 Effect of high glucose and Cr 3+ on TNF-α secretion in PBMCs isolated 

from normal human volunteers. Values represent mean ± SE (n = 4). Differences in values 

(* versus**, ** versus ##) are significant (p


HbA 1 (%) 

9 

8 

7 

6 

5 

4 

# 

## 

* 

+ 50 mM Glucose 

FIGURE 20.3 Effects of different chromium concentrations on hemoglobin glycation in 

high glucose-treated erythrocytes. Values represent mean ± SE (n = 4). Differences in 

values (# versus ##, ## versus **, ## versus ***, and ## versus ****) are significant (p 


Lipid Peroxidation 

(nmol malonidialdehyde/ml cells) 

1.2 

1.1 

1.0 

0.9 

0.8 

0.7 

0.6 

0.5 

* 

# 

* 

# 

FIGURE 20.4 Effect of chromium on lipid peroxidation in erythrocytes treated with 

different concentrations of glucose. Values represent mean ± SE (n = 4). Differences in 

values (* versus #) are significant (p


FIGURE 20.5 Proposed model for role of trivalent chromium supplementation in prevention 

of oxidative stress and vascular disease in diabetes. 

levels of pro-inflammatory cytokines and thereby improve insulin sensitivity and 

glycemic control among the diabetic patient population. If so, chromium supplementation 

may be used as an adjuvant therapy for reduction of vascular inflammation 

and CVD in diabetes. 


The author is supported by a grant from NIDDK and the Office of Dietary 

Supplements of the National Institutes of Health (RO1 DK064797), and thanks 

Georgia Morgan for excellent editing of this manuscript. 

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Monocyte-endothelial Cell Adhesion 

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Index 

A 

Abdominal obesity thresholds, 10 

gender comparisons, 10 

Adenosine triphosphate, uncoupling protein 2, 

pancreatic beta cell function 

beta cell mass, survival, 218–219 

insulin secretion, 216–218 

Adenosine triphosphate III 

initial report, metabolic syndrome 

definition, 5 

metabolic syndrome, 6 

revised criteria/guidelines, 7–9 

Adhesion molecules, clinical marker of 

inflammation, 152 

Adipokines, 116–118 

Adiponectin 

clinical marker of inflammation, 147–148 

oligomeric composition, 167–176 

adipocytes, 168–169 

central nervous system effects, 

172–173 

multimers, biological activities of, 

169–171 

thermogenesis, 172 

weight loss results, 171–172 

Adipose tissue, nutritional inflammation 

modulation, 231–232 

Alpha lipoic acid, 273–288. See also 

Lipoic acid 

adipocyte differentiation regulation, 280 

MAPK signaling pathways, 281–283 

transcription factor modulation, 

281–282 

diabetes mellitus prevention, 261–272 

hypothalamic AMPK, 277–279 

insulin resistance, 289–300 

etiology of insulin resistance, 292 

nutriceutical interventions, 292–295 

skeletal muscle glucose transport 

system, regulation, 291 

structure, 274 

American Association of Clinical 

Endocrinologists 

metabolic syndrome guidelines, 5–7 

Anti-inflammatory effect, insulin, proinflammatory 

effect, 

macronutrients, balance, 

metabolic syndrome, 15–32 

Antioxidants 

oxidative stress, 71–92 

childbirth, 75–78 

perinatal asphyxia, 76–77 

resuscitation, use of pure oxygen, 

77–78 

human milk, 82–84 

in overweight, obesity, 38–39 

perinatal period, 72–73 

pregnancy, 74–75 

fetal development, reactive oxygen 

species, 74 

preeclampsia, 75 

premature infants, 78–82 

bronchopulmonary dysplasia, 79 

neonatal necrotizing enterocolitis, 

80 

periventricular leukomalacia, 80–81 

retinopathy of prematurity, 81–82 

reactive oxygen species, tissueproduced, 

72 

perinatal period, oxidative stress, 71–92 

Atherosclerosis, inflammation, 139–166 

adhesion molecules, 152 

adiponectin, 147–148 

C-reactive protein, 144–146, 157–159 

cardiovascular risk, 141–143 

clinical markers, 143–152 

hemostatic factors, 159–160 

hemostatic parameters, 150–151 

interleukin-1, 152 



leptin, 146–147 

modification, 156–160 

resistin, 151–152 

tumor necrosis factor-alpha, 148–150, 159 

visfatin, 152 

Atherosclerotic plaque destabilization, lipidinduced 

macrophage death, 

251–260 

atherosclerosis, 254–255 

315


eicosanoids, 258 

programmed cell death, 253–254 

therapies, 258 

triacylglycerols, 255–257 

ATP. See Adenosine triphosphate 

B 

Beta cell failure, 115–116 

Body weight, obesity, 33–46 

abdominal thresholds, 10 

adipocyte differentiation, 273–288 

alpha lipoic acid, 261–272 

antioxidants, 38–39 



diabetes mellitus, 107–122, 261–272 

early life nutritional experience, correlation, 

62–63 

eicosapentaenoic acid, 41 

endothelial dysfunction, 261–272 

F 2-isoprostanes, 34–38 

fatty cell biology, 274–277 

hypothalamic AMPK suppression, 277–279 

increasing rates of, 108–110 

inflammation, 20–22, 94–96, 139–166 

insulin, macronutrient balance, 20–22 


International Diabetes Federation, 

abdominal threshold criteria, 10 

linoleic acid, 289–300 

lipoic acid, 273–300 

macronutrient, insulin balance, 20–22 

maternal, 93–106 

metabolic programming, 49–62 

nationality/gender comparisons, abdominal 

thresholds, 10 

nutrigenomics, 107–122 

oligomeric composition, adiponectin, 

167–176 

omega-3 polyunsaturated fatty acids, 39–41 


reduction, therapeutic targets, 38–41 

therapeutic targets for reduction, 38–41 

Bronchopulmonary dysplasia, 79 

C 

C-reactive protein, clinical marker of 

inflammation, 144–146, 157–159 

Cardiovascular disease, 111–113 

diabetes mellitus, post-prandial endothelial 

dysfunction 

fasting hyperglycemia, 124–125 

hyperglycemic spikes, 124–127 

mechanisms, 127–128 

oxidative, nitrosative stress, 128–131 

post-prandial hyperglycemia, 

128–131 

post-prandial hyperglycemia, 124–127 

nutritional inflammation modulation, 

228–229 

Central nervous system effects, adiponectin, 

172–173 

Childbirth, oxidative stress, 75–78 


resuscitation, use of pure oxygen, 77–78 

Chinese national origin, abdominal obesity 


Chromium supplementation, 301–312 

diabetes mellitus, 303–307 

oxidative stress, 303 

pro-inflammatory cytokines 


vascular inflammation, 302–303 

vascular inflammation, 303 

CLA. See Conjugated linoleic acid 

Clinical markers, inflammation, 143–152, 

156–160 




hemostatic parameters, 150–151 




leptin, 146–147 


tumor necrosis factor-alpha, 148–150, 159 

visfatin, 152 

Conjugated linoleic acid, insulin resistance, 

289–300 



skeletal muscle glucose transport system, 

regulation, 291 

D 

Definition of metabolic syndrome, 3–14 

abdominal obesity thresholds, 10 

adenosine triphosphate III 

initial report, 5 

metabolic syndrome, 6

Index 317 

revised criteria/guidelines, 7–9 

American Association of Clinical 

Endocrinologists, 5–7 

European Group for Study of Insulin 

Resistance Syndrome, 7–8, 12 

features of metabolic syndrome definitions, 

12 

INTERHEART project, 3, 9 

International Diabetes Federation 

definition, 8–9 

metabolic syndrome criteria, 10 

metabolic syndrome definition, 12 


obesity thresholds, 10 

rationale for defining, 3–5 

World Health Organization 

metabolic syndrome definition, 12 

metabolic syndrome diagnostic criteria, 

6 

World Health Organization definition, 5 

Diabetes mellitus 


nutrigenomics, metabolic syndrome, 

107–122 

adipokines, 116–118 

beta cell failure, 115–116 

cardiometabolic risk, 111–113 

criteria for diagnosing metabolic 

syndrome, 113 

patient assessment, 109 

pre-diabetes mellitus, 111–113 

nutritional inflammation modulation, 229 

post-prandial endothelial dysfunction, 

123–138 

cardiovascular disease 




oxidative, nitrosative stress, 

128–131 


128–131 


124–127 

trivalent chromium supplementation, 

302–307 

vascular remodeling, 195–210 

Akt/PKB signaling pathway, 202 

dual PPAR activators, 204 

e-Jun N-terminal kinase signaling, 201 

extracellular signal-regulated kinase 1 

signaling, 199–200 

inflammation, vascular, 203–204 

MAPK signaling, 198 

p38 signaling, 200–201 

PI3K signaling pathway, 201–202 

PPAR-gamma, 199–200 

reactive oxygen species, 202–203 

Dietary fatty acids, metabolic syndrome, 

inflammation, 243–250 

Dihydrolipoic acid, structure, 262, 274 

Dysplasia, bronchopulmonary, 79 

E 

Early life nutritional modifications, metabolic 

syndrome, 47–70 


generational effects, 59–62 

high carbohydrate model, 54–58 

immediate effects, 56–58 

immediate post-natal period, 53–54 

persistent effects in adulthood, 58–59 

pre-natal metabolic programming, 

50–53 

EGIR syndrome. See European Group for Study 

of Insulin Resistance Syndrome 

Eicosapentaenoic acid, oxidative stress, in 

overweight, obesity, 41 

Energy expenditure 



MAPK signaling pathways, 

281–283 


281–282 


Enterocolitis, neonatal, necrotizing, 80 

EPA. See Eicosapentaenoic acid 

European Group for Study of Insulin Resistance 

Syndrome, 7–8, 12 

European national origin, abdominal obesity 


F 

F 2-isoprostanes, oxidative stress in overweight, 

obesity, 34–38 

Fasting hyperglycemia, cardiovascular disease, 

124–125 

Fatty acids, metabolic syndrome, inflammation, 

243–250 

Features of metabolic syndrome definitions, 12


G 

Gender comparisons, abdominal obesity 


Generational effects, early life nutritional 

modifications, 59–62 

Glucose intolerance, maternal obesity, 

pregnancy, inflammation, 93–106 

fetal growth, 100 

interventions, 101–103 

H 

High carbohydrate model, early life nutritional 

modifications, 54–58 

Human islets, UCP2 protein expression, 213 

Hyperglycemic spikes, cardiovascular disease, 

124–127 

Hypertension, vascular remodeling, 195–210 














Hypocaloric diets, inflammation modulation, 

232–237 

I 

IDF. See International Diabetes Federation 

Inflammation 

in atherosclerosis, 139–166 

dietary fatty acids, insulin signaling, 

244–246 

insulin, anti-inflammatory effect, 15–32 

metabolic syndrome, 15–32, 227–242 

in pregnancy, 93–106 

pro-inflammatory effect, macronutrients, 

15–32 


301–312 

in type 2 diabetes, 123–138 

vascular, 203–204, 301–312 

Insulin resistance, 16, 289–300 


inflammatory hypothesis, 22 

macronutrient, metabolic effects, 16–17 

metabolic effects, 16–17 


skeletal muscle glucose transport system, 


Insulin resistance syndrome, usage of term, 7 

Insulin signaling, dietary fatty acids, 243–250 

Insulin-stimulated reactive oxygen species, 

signal transduction, 177–194 

cellular insulin stimulation, H 2O 2 

generation, 181 

cellular tyrosine kinase signaling, reactive 

oxygen species as second 

messengers, 179 

insulin-stimulated H 2O 2 generation, proteintyrosine 

phosphatase, 1B 


novel targets, 186 

Nox4, signaling regulation, insulin action 

cascade, 184–185 

protein-tyrosine phosphatase, 180–181 

reversible tyrosine phosphorylation, 

178–179 

INTERHEART project, 3, 9 

Interleukin-1, clinical marker of inflammation, 

152 


152 


152 

International Diabetes Federation 

metabolic syndrome, 12 

criteria, 10 

definition, 8–9 

International Diabetes Federation criteria, 

thresholds for abdominal obesity, 

10 

J 

Japanese national origin, abdominal obesity 


L 

Leptin, clinical marker of inflammation, 

146–147 

Leukomalacia, periventricular, 80–81

Index 319 

Lipid-induced macrophage death, 

atherosclerotic plaque 

destabilization, 251–260 


eicosanoids, 258 

programmed cell death, 253–254 

therapies, 258 

triacylglycerols, 255–257 

Lipoic acid, 273–288. See also alpha lipoic acid 


MAPK signaling pathways, 281–283 


281–282 





skeletal muscle glucose transport 

system, regulation, 291 

structure, 262 

M 

Macronutrients, insulin balance, metabolic 



insulin resistance, 16 

inflammatory hypothesis, 22 

metabolic effects, 16–17 

non-metabolic actions of insulin, 17–20 


origin of inflammation, 22–25 

pathogenesis, 24 

MAPK signaling pathways, responses, 198 

Maternal obesity, 93–106 

glucose intolerance, inflammation, 93–106 



Metabolic syndrome, 107–122. See also 

diabetes mellitus 

criteria for diagnosing, 113 

definition of, 3–14 .See also under 

Definition of metabolic syndrome 

dietary fatty acids, 243–250 .See also under 

Dietary fatty acids 

inflammation modulation, 227–242 .See 

also under Inflammation 

macronutrients, insulin balance, 15–32 .See 

also under Macronutrients, insulin 

balance, metabolic syndrome 

nutrigenomics, diabetes mellitus, 107–122 




criteria for diagnosing metabolic 

syndrome, 113 



nutritional modifications, early life, 47–70. 

See also Nutritional modifications, 

early life 

Mitogen-activated protein kinase. See MAPK 

N 

Nationality/gender comparisons, abdominal 

obesity thresholds, 10 

Neonatal necrotizing enterocolitis, 80 

Non-metabolic actions of insulin, 17–20 

Nutrigenomics, diabetes mellitus, metabolic 





criteria for diagnosing metabolic syndrome, 

113 



Nutritional modifications, early life, metabolic 



generational effects, 59–62 

high carbohydrate model, 54–58 

immediate effects, 56–58 

immediate post-natal period, 53–54 

persistent effects in adulthood, 58–59 

pre-natal metabolic programming, 

50–53 

O 

Obesity, 20–22, 33–46 

abdominal thresholds, 10 

adipocyte differentiation, 273–288 








clinical markers, inflammation, 143–152 

hemostatic factors, inflammation 

modification, 159–160 

hemostatic parameters, 150–151





leptin, 146–147 

modification of inflammation, 156–160 


tumor necrosis factor-alpha, 148–150, 

159 

visfatin, 152 


diabetes mellitus, 107–122, 261–272 

early life nutritional experience, correlation, 

62–63 

early life nutritional modifications, 47–70 


endothelial dysfunction, 261–272 


fatty cell biology, 274–277 

hypothalamic AMPK suppression, 277–279 

increasing rates of, 108–110 

inflammation, 20–22, 94–96, 139–166 

insulin, macronutrient balance, 20–22 


International Diabetes Federation, 

abdominal threshold criteria, 10 

linoleic acid, 289–300 

lipoic acid, 273–300 

macronutrient, insulin balance, 20–22 

maternal, 93–106 




nutrigenomics, 107–122 

oligomeric composition, adiponectin, 

167–176 




therapeutic targets for reduction, 38–41 

Oligomeric composition, adiponectin, 167–176 

adipocytes, 168–169 

central nervous system effects, 172–173 

multimers, biological activities of, 169–171 

thermogenesis, 172 

weight loss results, 171–172 

Omega-3 polyunsaturated fatty acids, oxidative 

stress, in overweight, obesity, 

39–41 

Origin of inflammation, 22–25 

Oxidative stress 


childbirth, 75–78 


P 

resuscitation, use of pure oxygen, 

77–78 

human milk, 82–84 


pregnancy, 74–75 


species, 74 


premature infants, 78–82 


neonatal necrotizing enterocolitis, 

80 



reactive oxygen species, tissueproduced, 

72 






as markers, 34–35 

omega-3 polyunsaturated fatty acids, 

39–41 



trivalent chromium supplementation, 303 

Pancreatic beta cell function, uncoupling 

protein 2, 211–224 

adenosine triphosphate 



expression, regulatory factors of, 212–216 

reactive oxygen species production, beta cell 

mass, survival, 218–219 

Perinatal asphyxia, 76–77 

Perinatal period, oxidative stress, 71–92 

Periventricular leukomalacia, 80–81 

Peroxisome proliferator-activated receptors, 

hypertension, 195–210 

Post-natal period metabolic programming, 

53–54 

Post-prandial endothelial dysfunction, diabetes 

mellitus, 123–138 

cardiovascular disease 




oxidative, nitrosative stress, 128–131

Index 321 


128–131 

post-prandial hyperglycemia, 124–127 

PPARs. See Peroxisome proliferator-activated 

receptors 

Pre-natal metabolic programming, 50–53 

Preeclampsia, oxidative stress, 75 

Pregnancy, 93–106 

glucose intolerance, inflammation, 93–106 



maternal obesity, glucose intolerance, 






species, 74 


Premature infants, oxidative stress, 78–82 


neonatal necrotizing enterocolitis, 80 



Pro-inflammatory cytokines 

monocytes, inflammation modulation, 

230–231 

trivalent chromium supplementation 



vascular inflammation, trivalent chromium 

supplementation, 302–303 

Pro-inflammatory effect, macronutrients, antiinflammatory 

effect, insulin, 

balance, metabolic syndrome, 

15–32 

R 

Rationale for defining metabolic syndrome, 3–5 

Resistin, clinical marker of inflammation, 

151–152 

Resuscitation in perinatal asphyxia, use of pure 

oxygen, 77–78 

Retinopathy of prematurity, 81–82 

S 

Signal transduction, insulin-stimulated reactive 

oxygen species, 177–194 

cellular insulin stimulation, H 2O 2 

generation, 181 

cellular tyrosine kinase signaling, reactive 

oxygen species as second 

messengers, 179 

insulin-stimulated H 2O 2 generation, proteintyrosine 

phosphatase, 1B 


novel targets, 186 

Nox4, signaling regulation, insulin action 

cascade, 184–185 

protein-tyrosine phosphatase, 180–181 

inhibition, 183 

reversible tyrosine phosphorylation, 

178–179 

South Asian national origin, abdominal obesity 


Stress, oxidative, in overweight, obesity, 33–46 



F 2-isoprostane formation, 35 


as markers, 34–35 



T 

Thermogenesis, adiponectin, 172 

Trivalent chromium supplementation, 301–312 


oxidative stress, 303 

pro-inflammatory cytokines 



vascular inflammation, 303 

Tumor necrosis factor-alpha, clinical marker of 

inflammation, 148–150, 159 

U 

UCP2 protein expression, human islets, 213 

Uncoupling protein 2 

pancreatic beta cell function, 211–224 

adenosine triphosphate 



expression, regulatory factors of, 

212–216 

reactive oxygen species production, beta 

cell mass, survival, 218–219


V 

Vascular inflammation 

pro-inflammatory cytokines, trivalent 

chromium supplementation, 

302–303 


302–303 

Vascular remodeling, hypertension, diabetes 

mellitus, 195–210 














Visfatin, clinical marker of inflammation, 152 

W 

WHO. See World Health Organization 

World Health Organization 

metabolic syndrome 

definition, 12 

diagnostic criteria, 6 

metabolic syndrome definition, 5

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